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CELLULAR AND MOLECULAR

Ca²⁺ Channel Blockers Enhance Neurotensin (NT) Binding and Inhibit NT-Induced Inositol Phosphate Formation in Prostate Cancer PC3 Cells

Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts (R.E.C., X.G.); and Department of Biology, Tufts University, Medford, Massachusetts (D.E.C.)

Received for publication April 4, 2003
Accepted July 8, 2003.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Neurotensin (NT) stimulates Ca²⁺ release and Ca²⁺ influx in many cells. Its contractile effects in smooth muscle are inhibited by removal of Ca²⁺ and by Ca²⁺ channel blockers (CCBs). To better understand NT signaling in prostate cancer PC3 cells, blockers of voltage-gated and store-operated Ca²⁺ channels (VGCC and SOCC) were tested for effects on NT-binding and signaling. Eight chemical types of agents, including VGCC-blocker nifedipine and SOCC-blocker SKF-96365 (1-[-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenyl]-1H-imidazole), enhanced cellular NT binding up to 3-fold, while inhibiting (by 70%) NT-induced inositol phosphate (IP) formation. The ability to enhance NT binding correlated with the ability to inhibit NT-induced IP formation, and both effects were relatively specific for NT. Although cellular binding for ₂-adrenergic, V_1a-vasopressin, and epidermal growth factor receptors was not enhanced by these drugs, bombesin receptor binding was increased 19% and bombesin-induced IP formation was inhibited 15%. One difference was that the effect on NT binding was Ca²⁺-independent, whereas the effect on IP formation was Ca²⁺-dependent (in part). The Ca²⁺-dependent part of the IP response seemed to involve SOCC-mediated Ca²⁺ influx to activate phospholipase C (PLC), while the Ca²⁺-independent part probably involved PLC. Photoaffinity labeling of the NT receptor NTR1 was enhanced in CCB-treated cells. NTR1 affinity was increased but NTR1 number and internalization were unchanged. Since CCBs did not alter NT binding to isolated cell membranes, the effects in live cells were indirect. These results suggest that CCBs exert two effects: 1) they inhibit NT-induced IP formation, perhaps by preventing Ca²⁺ influx-dependent activation of PLC; and 2) they enhance NTR1 affinity by an unexplained Ca²⁺-independent mechanism.

Some actions of NT depend on extracellular [Ca²⁺] and are inhibited by Ca²⁺ channel blockers (CCBs). Based on the effects of 1,4-dihydropyridines (DHPs) such as nifedipine (NIF), Donoso et al. (1986) and Mule and Serio (1997) suggested that NT-induced intestinal contraction involved Ca²⁺ influx through voltage-gated Ca²⁺ channels (VGCC). However, in some systems, VGCC current is inhibited by NT (Belmeguenai et al., 2002), and DHPs inhibit NT effects independently of Ca²⁺ (Golba et al., 1995). These contradictory findings led us to investigate the effects of CCBs on NT binding and signaling in prostate cancer PC3 cells, which express functional NTR1 (Seethalakshmi et al., 1997). We hypothesized that CCBs could exert effects at multiple levels of the signaling pathway.

Our studies in PC3 cells indicate that NTR1 is linked to G_q/11 and that stimulation by NT activates PLC, enhances IP formation, and elevates cellular [Ca²⁺]. This signaling pathway contributes to the regulation of cellular growth by NT (Seethalakshmi et al., 1997) and is linked to a conditional activation of adenylyl cyclase (Carraway and Mitra, 1998). Ca²⁺ is required for NT to stimulate DNA synthesis and to enhance cAMP formation, and these effects are inhibited by the VGCC blocker NIF (R. E. Carraway, unpublished results). Although this suggests that Ca²⁺ influx participates in NT signaling, the roles of Ca²⁺ channels, Na⁺/Ca²⁺ exchange, and Ca²⁺ pumps are not defined. The inhibitory effects of NIF implicate VGCC in NT signaling, but this must be questioned since PC3 cells are epithelial and nonexcitable (Putney and Bird, 1993). Another process, which occurs in excitable and nonexcitable cells (Parekh, 2003) subsequent to Ca²⁺ store emptying (Putney, 1999), is capacitative Ca²⁺ entry through store-operated Ca²⁺ channels (SOCC). Given that NT stimulates capacitative Ca²⁺ entry in Chinese hamster ovary cells (Gailly, 1998) and that NT elevates cellular [Ca²⁺] in PC3 cells (R. E.Carraway, unpublished results), it seems likely that NT stimulates SOCC-mediated Ca²⁺ influx in PC3 cells. Thus, additional studies are required to determine whether NT induces Ca²⁺ influx and to define the channels and mechanisms involved.

Our ability to distinguish mechanisms of Ca²⁺ entry depends largely on the selectivity of CCBs (Harper and Daly, 1999; Triggle, 1999). Based on its possible relevance to NT signaling as discussed above, we focus the following discussion on blockers of L-type VGCC and SOCC. Blockers of VGCC include DHPs (e.g., NIF), phenylalkylamines (e.g., verapamil), and benzothiazepines (e.g., diltiazem). Inhibitors of SOCC include imidazoles (e.g., SKF-96365) and tricyclics (e.g., trifluoperazine). Unfortunately, these agents exhibit some nonspecificity, and their actions can be complex (Harper et al., 2003; Triggle, 2003). Although selective for VGCC at nanomolar levels, DHPs inhibit capacitive Ca²⁺ entry in the micromolar range (Harper et al., 2003). Ligand-gated ion channels are also targets of DHPs. Ca²⁺ influx involving the N-methyl-D-aspartate receptor was inhibited by 1 to 10 µM nitrendipine (Skeen et al., 1993), nicotinic acetylcholine receptor-induced currents were abolished by 10 µM NIF (Lopez et al., 1993), and the 5-hydroxytryptamine receptor was inhibited by 10 µM nimodipine (Hargreaves et al., 1996). These findings attest to the need to examine multiple agents and to assess the effects on each step leading to downstream events. Yet, in performing experiments to order signaling steps, it is often assumed that the agents tested (e.g., CCBs) do not alter the agonist-receptor interaction. Even when this is examined, cellular membranes are commonly used, which provides a limited assessment. These considerations have led us to focus attention on the early steps in the signaling pathway, NT binding, and NT-induced IP formation in live cells.

The current study investigates the effects of CCBs on NT binding and signal transduction in PC3 cells. Screening ion channel agents for effects on NT signaling, we unexpectedly find that VGCC and SOCC blockers dramatically enhance NT binding and cause a parallel inhibition of NT-induced IP formation. We document the specificity of these effects in regard to agent and receptor, studying eight classes of CCBs and five receptors. We find that the efficacy order to enhance NT binding is similar to that for inhibition of IP production, and these effects display similar receptor selectivity. Detailed studies examine the effects on NTR1 and investigate the involvement of Ca²⁺-dependent PLC(s).

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. The radiochemicals, [¹²⁵I]sodium iodide (2000 Ci/mmol), [1,2-³H(N)]myoinositol (60 mCi/mmol), and [⁴⁵Ca]calcium chloride (>10 Ci/g) were obtained from PerkinElmer Life Sciences (Boston, MA). Phloretin, 2-aminoethoxydiphenylborate (2-APB), tetrandrine, human EGF, -conotoxin, ionomycin, thapsigargin, and veratridine were purchased from Calbiochem (San Diego, CA). Glibenclamide, diazoxide, ryanodine, dantrolene, and 1-[-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenyl]-1H-imidazole (SKF-96365) were obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). Des-Gly-[Phaa¹,D-Tyr(Et)²,Lys⁶,Arg⁸]vasopressin (HOLVA) was purchased from Peninsula Laboratories (Belmont, CA). Nimodipine, verapamil, diltiazem, NT, NIF, miconazole, tetraethylammonium, flunarizine, phenylarsine oxide, amiloride, pindolol, and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). [4-azido-Phe⁶]NT was synthesized using reagents obtained from Novabiochem (San Diego, CA). SR48692 was generously provided by Danielle Gully at Sanofi-Synthelabo (Toulouse, France). Stocks of test agents were prepared daily (10 mM in DMSO) and diluted into Locke's solution just before use, except for SKF-96365, miconazole, and trifluoperazine (dissolved in Locke's solution).

Iodinations. Iodinations of ligands (EGF, 3 nmol; all others, 15 nmol) were performed using chloramine T (10 µg) as described (Carraway et al., 1993). All reactions were stopped using sodium metabisulfite (30 µg) except for EGF (stopped by dilution). The monoiodinated products were purified by reverse-phase HPLC using µBondapak-C18 (3.9 x 300 mm column) eluted at 1.5 ml/min with a linear gradient (60 min) from 0.1% trifluoroacetic acid to 60% CH₃CN. The specific activity of the purified ¹²⁵I-NT was 1000 to 2000 cpm/fmol as determined by radioimmunoassay (Carraway et al., 1993).

Binding to PC3 Cells. PC3 cells, obtained from the American Type Culture Collection (Manassas, VA) were maintained (passage 4) by our tissue culture facility at the University of Massachusetts Medical School (Seethalakshmi et al., 1997). Cells, passaged no more than 30 times, were grown to 95% confluency in 24-well culture plates. For binding studies, cells were washed with 2 ml/well Hepes-buffered Locke-BSA (Locke) (148 mM NaCl, 5.6 mM KCl, 6.3 mM Hepes, 2.4 mM NaHCO_3, 1.0 mM CaCl_2, 0.8 mM MgCl_2, 5.6 mM glucose, 0.1% BSA; pH 7.4). Equilibrium binding at 37°C was performed for 25 min using 10⁵ cpm/ml of each ¹²⁵I-labeled ligand in 1.0 ml Locke with varying amounts of unlabeled ligand (0-1 µM). 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. During this 5-min washing procedure dissociation of ¹²⁵I-NT from cell receptors was <1%. Total cellular binding was assessed by measuring radioactivity and protein (Bio-Rad assay; BSA standard) in cells extracted in 0.6 ml of 0.2 M NaOH. A Packard 10-well gamma counter was used to measure radioactivity. Specific binding was defined as that displaceable by 1 µM ligand.

Cell surface binding and internalization of ¹²⁵I-NT were assessed by washing cells at 22°C for 2 min with 0.6 ml of 0.2 M acetic acid, 0.5 M NaCl, pH 3.0 (Beaudet et al., 1994). Binding at 4°C achieved equilibrium within 3 h, at which time >90% of the radioactivity was on the cell surface. Binding at 37°C reached equilibrium in 25 min, at which time 70% of total binding was internalized. To measure rates of internalization for ¹²⁵I-NT prebound to cells, the following procedure was used. ¹²⁵I-NT (10⁵ cpm) was prebound to PC3 cells in 24-well plates at 4°C for 3 h. After washing the cells three times in ice-cold phosphate-buffered saline (PBS), >90% of ¹²⁵I-NT was located on the cell surface as determined by acid washing. Agents (10 mM in DMSO) were diluted to 50 µM in Locke and incubated with the cells at 37°C for 5 min. The control was 0.5% DMSO. Cell-surface and internalized ¹²⁵I-NT were measured, and the percentage of internalization per minute was calculated.

Binding displacement curves were constructed for each set of treatments and binding parameters were determined by Scatchard analysis. The K_i value was determined by using the equation K_i = IC₅₀/1 + [L]/K_d, where K_d and [L] are the dissociation constant and the concentration of the ligand, respectively (Cheng and Prusoff, 1973).

Assessment of Binding Assay Artifacts. CCBs did not alter the ability of cells to adhere to plates, as evidenced by protein assay. Typically, each well contained 188 ± 11 µg (control, n = 6), 183 ± 10 µg (50 µM NIF, n = 6), 190 ± 12 µg (50 µM phloretin, n = 6), and 181 ± 11 µg of protein (100 µM verapamil, n = 6) after binding and washing.

CCBs did not alter the stability of ¹²⁵I-NT during binding at 37°C. After binding at 37°C, >90% of the radioactivity in the medium eluted during HPLC at the position of ¹²⁵I-NT for cells incubated in buffer, 50 µM NIF, or 100 µM verapamil. HPLC was performed at 1.5 ml/min on µBondapak-C18 (8 x 100 mm) with linear gradient (20 min) from 0.1% trifluoroacetic acid to 30% CH₃CN. ¹²⁵I-NT eluted at 25.0 min. During binding at 37°C, 4% of the added ¹²⁵I-NT was bound to the cells. Therefore, the medium was sampled in time and tested for the ability to bind to fresh cells. The loss of binding ability was 5% after 25 min. The protease inhibitors o-phenanthroline and PMSF (0.5 mM), had no effects on HPLC profiles and on loss of binding ability over time.

CCBs did not alter the dissociation rate of ¹²⁵I-NT from cellular receptors during washing with ice-cold saline. When cells were labeled with ¹²⁵I-NT in buffer, 50 µM NIF, or 100 µM verapamil, dissociation of cell-associated radioactivity was negligible (<6%) during incubation in ice-cold saline for 15 min.

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. Membranes were collected and washed onto glass fiber (GF-B) filters using a Brandel cell harvester, and the filters were counted (Seethalakshmi et al., 1997).

Cross-Linking of ¹²⁵I-[4-azido-Phe⁶]-NT to NTR1. [4-azido-Phe⁶]-NT was iodinated and purified by HPLC to 1500 Ci/mmol. PC3 cells in 10-cm dishes were incubated with 0.3 x 10⁶ cpm/ml ¹²⁵I-[4-azido-Phe⁶]-NT in 8 ml of Locke for 25 min at 37°C in the presence and absence of Ca²⁺ channel agents; 1 µM NT was added to controls. Cells were placed on ice for 30 min, irradiated at 254 nm with a hand-held UV light for 5 min at 3 cm, washed in ice-cold PBS, and lysed in 10 mM Hepes, 1 mM EDTA, 0.5 mM o-phenanthroline, PMSF, and N-tosyl-L-phenylalanine chloromethyl ketone (pH 7.4). Membranes, collected by centrifugation (30,000g, 20 min) were solubilized in 250 µl of 50 mM Tris buffer (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, 0.5% NP-40, and 5% glycerol at 4°C for 2 h. Solubilized NTR1, diluted 2-fold in buffer without detergent, was immunoprecipitated by addition of our rabbit antiserum (Ab-NTR1) toward the C-terminal 15 residues of human NTR1 (final 1:100). During Western blotting, Ab-NTR1 detected two major bands in extracts of PC3 cells, the parent protein of 50 kDa and a breakdown product of 33 kDa, in keeping with results in other cells (Boudin et al., 1995). After 18 h at 4°C, protein A-agarose (10 mg; Sigma-Aldrich) was added for 6 h. After the agarose beads were washed three times with PBS at 4°C, associated radioactivity was measured using a gamma counter. Usually the immunoprecipitate contained 5% of the solubilized cpm for samples prepared in the absence of NT. SDS-PAGE was used in some cases to validate that the radiolabeled material represented NTR1. For this, the beads were boiled for 5 min in an equal volume of 2x SDS sample buffer, and extracts were subjected to SDS-PAGE using 10% polyacrylamide gels, followed by autoradiography.

Western Blotting. PC3 cells in 60-mm dishes were washed in Locke containing inhibitors 0.5 mM EDTA, 0.5 µM PMSF, 0.5 µM N-tosyl-L-phenylalanine chloromethyl ketone, and 0.5 µM o-phenanthroline. Cells were lysed in 100 µl of 2x SDS loading buffer with inhibitors, scraped into microfuge tubes, and sonicated (20 s) on ice. Membranes were isolated from regions of adult rat brains (Carraway et al., 1993), and P2 pellets were extracted in 2x SDS loading buffer and sonicated. Cell and tissue extracts were boiled 5 min and separated by SDS-PAGE on 10% polyacrylamide minigels. Proteins were electroeluted onto polyvinylidene difluoride (Immobilon P; Millipore Corporation, Bedford, MA). After blocking in 5% nonfat milk in TTBS (0.05% Tween 20, 20 mM Tris, 0.5 M NaCl) for 1 h and washing 3 times with TTBS, blots were incubated with the primary antiserum (1:1000) in blocking buffer for 18 h at 4°C. Our rabbit antiserum (Ab-NTR1) was raised using a synthetic peptide corresponding to residues 398-418 of human NTR1 conjugated to keyhole limpet hemocyanin. The antibodies were affinity-purified before use. Blots were washed in TTBS, then incubated with horseradish peroxidase-linked goat anti-rabbit antibody (1:1000) for1hat20°C, and washed again in TTBS. Enhanced chemiluminescence was performed according to the manufacturer's instructions (Santa Cruz Biochemicals, Santa Cruz, CA). After stripping (1 h at 70°C in 62.5 mM Tris-HCl, 2% SDS, 0.1 M -mercaptoethanol, pH 6.8) and washing in TBS, blots were reprobed with antigen-adsorbed antisera to validate the results.

Influx of ⁴⁵Ca²⁺ into PC3 Cells. The method of Katsura et al. (2000) was used to measure ⁴⁵Ca²⁺ influx in response to NT. Briefly, confluent PC3 cells in 24-well dishes were washed with Ca²⁺-free Locke and pretreated for 10 min with 0 to 36 µM NIF (600 µl/well). The reaction was initiated by the addition of 200 µl of NT followed in 2 min by 2.5 mM CaCl₂ (5 µCi ⁴⁵Ca²⁺/well). After 8 min, the cells were washed three times with ice-cold Locke and solubilized in 1.0 ml of 0.25 M NaOH. The cell extract was neutralized with acetic acid, and an aliquot was subjected to liquid scintillation spectrometry to measure ⁴⁵Ca²⁺ radioactivity.

Measurement of IP Formation. IP formation was measured by the method of Chen and Chen (1999) wherein [³H]inositol was used to label the phosphoinositide pool. PC3 cells in 24-well plates were incubated 48 h with myo-[³H]inositol (2.5 µCi/ml) in medium 199 and 5% fetal calf serum. Medium 199 (Difco, Detroit, MI) was chosen because of its low inositol content. After washing with 2 ml of Locke, cells were preincubated 10 min with test agents in Locke and 15 mM LiCl, and reactions were started by adding a maximal dose of NT (30 nM) or vehicle. After 30 min at 37°C, the 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 transferred to tubes using 2x 2-ml water washes, and [³H]IP was adsorbed to 0.25 ml AG-1 x 8 slurry (formate form; Bio-Rad, Hercules, CA), which was washed five times in 5 mM myoinositol (5 ml) and eluted in 0.75 ml of 1.5 M ammonium formate and 0.1 M formic acid. Scintillation counting was performed on 0.5 ml of eluate in 5 ml of Ecoscint (National Diagnostics, Atlanta, GA). For experiments involving removal of Ca²⁺ from the buffer, cells were washed with Ca²⁺-free buffer and used immediately to minimize any disturbance to internal Ca²⁺ stores.

Statistics. Statistical comparisons were made using the Student's t test. Results were calculated as mean ± S.E.M. and p < 0.05 was considered significant.

	Results

Top Abstract Materials and Methods Results Discussion References

 
CCBs Enhanced Cellular Binding of NT. Specific binding of ¹²⁵I-NT (10⁵ cpm/ml) to PC3 cells at 37°C was >95% of total binding and was 16.8 ± 0.81 cpm ¹²⁵I-NT bound/µg protein (n = 12), which corresponded to 3000 cpm ¹²⁵I-NT bound per well. Table 1 gives the binding parameters determined for NT binding to PC3 cells. Data given under Materials and Methods attests to the validity of the assay, showing that the ¹²⁵I-NT remained intact during incubation and that dissociation did not occur during washing. CCBs did not alter these conditions.

CCBs (e.g., NIF, phloretin, and verapamil) increased the apparent rate¹ of and the steady-state level of NT binding to PC3 cells (Fig. 1A). NIF enhanced specific binding without altering nonspecific binding, and it was effective across a 10-fold range in cell density (Fig. 1B). Similar effects were displayed by five L-type VGCC blockers, two L-type/T-type VGCC blockers, and two blockers of SOCC, representing seven different classes of chemicals. The order of efficacy (NIF > phloretin > verapamil > diltiazem) for VGCC blockers was similar to that for peripheral vasodilation (Triggle, 1999). NT binding was increased up to 3.1-fold by NIF, 2.9-fold by phloretin, 2.0-fold by verapamil, and 1.4-fold by diltiazem (Fig. 1C). Nimodipine and NIF were the most potent agents, elevating NT binding at submicromolar concentrations [control, 100 ± 4%; 0.3 µM nimodipine, 116 ± 5% (p < 0.05); 0.9 µM NIF, 115 ± 5% (p < 0.05)]. Although less specific CCBs (flunarizine, tetrandrine, trifluoperazine, and chlorpromazine) had only modest effects (Table 2), well defined blockers of SOCC (SKF-96365, miconazole) enhanced NT-binding up to 2.9-fold (Fig. 1D; Table 2).

View larger version (34K): [in this window] [in a new window]   Fig. 1. CCBs increased the rate and steady-state level of NT binding to intact PC3 cells. A, PC3 cells were preincubated with 30 µM NIF, phloretin, or verapamil or with 0.3% DMSO at 37°C. After 10 min, 125I-NT (105 cpm; 50 pM final) was added, along with NT (1 µM final) or control. Incubation continued for times indicated, reactions were stopped, and binding was expressed as cpm/µg protein. Results are for n = 3 in a typical experiment that was repeated. B, cells, diluted 1:1, 1:3, and 1:9, were plated, yielding 200 µg, 75 µg, and 20 µg of protein/well, respectively. Cells were preincubated 10 min with 30 µM NIF or vehicle, and NT binding (37°C, 25 min) was measured. Results are n = 6 in a typical experiment performed three times. C and D, cells were pretreated 10 min with indicated agents and NT binding was measured. The minimum dose that significantly (p < 0.05) elevated NT binding above control was 0.3 µM (nimodipine), 0.9 µM (NIF), 20 µM (verapamil), 10 µM (phloretin), 4 µM (SKF-96365), 10 µM (miconazole), and 100 µM (diltiazem). Results are from four experiments.

CCBs Inhibited NT-Induced IP Formation. NT increased IP formation 5-fold in PC3 cells with an EC₅₀ value of 1 nM (Fig. 2A). L-type VGCC blockers inhibited the response to a maximal dose of NT (Fig. 2B), with an efficacy order (NIF > phloretin > verapamil) similar to that for enhancement of NT binding (Table 2). SOCC blockers also inhibited the response to NT (Fig. 2B), giving an efficacy order (SKF-96365 > miconazole > trifluoperazine) similar to that for enhancing NT binding (Table 2). For each of these agents the EC₅₀ value for enhancing NT binding was similar to the IC₅₀ value for inhibiting NT-induced IP formation (Table 2), and there was a strong statistical correlation (r² = 0.842). These results indicated that the drug effects on NT binding and NT-induced IP formation had a similar chemical sensitivity and/or that the two effects were linked, e.g., that one led to the other.

View larger version (23K): [in this window] [in a new window]   Fig. 2. NT-induced IP formation (A) was inhibited by CCBs (B). A, log dose-response plot showing that NT enhanced IP formation 4.5-fold with EC50 1.0 nM. The minimum dose of NT that significantly (p < 0.05) elevated IP formation above control was 0.2 nM. B, log dose-response plots showing that IP formation in response to a maximal dose of NT (30 nM) was inhibited by CCBs. The minimum dose that significantly (p < 0.05) decreased IP formation below control was 5 µM (NIF), 7 µM (phloretin), 7 µM (SKF-96365), 20 µM (miconazole), and 40 µM (verapamil). Results are from 10 experiments (A) and 3 to 4 experiments (B).

Tyrosine Kinase Inhibitors Increased ¹²⁵I-EGF Binding to PC3 Cells. Tyrosine kinase inhibitors have been identified that specifically bind to the ATP binding site of EGFR and block kinase function (Arteaga et al., 1997). Since these drugs were known to greatly elevate EGF binding in some cancer cells (Lichtner et al., 2001), we tested their effects in PC3 cells. Initially, we showed that the PC3 cell surface displayed EGFR with high affinity for ¹²⁵I-EGF (Table 1). Testing the effects of tyrosine kinase inhibitors, we found that AG1478 and PD153035 increased EGF binding to PC3 cells by as much as 4.3-fold (Fig. 3A), whereas they had little effect on NT binding (Fig. 3B). In contrast, CCBs NIF and SKF-96365 had little effect on EGF binding (Fig. 3A, Table 5), although they enhanced NT binding 3-fold (Figs. 1, C and D, and 3B). These results not only demonstrated specificity, but also a degree of similarity to these systems, since the elevated binding in both cases was associated with an inhibition of the response to receptor activation.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Tyrosine kinase inhibitors increased 125I-EGF binding to PC3 cells (A) but had little effect on NT binding (B). PC3 cells were preincubated 10 min with indicated agents, and binding was performed at 37°C. Results from five experiments (A) and three experiments (B) were expressed as percentage of control and plotted as mean ± S.E.M. A, whereas EGF binding was not altered by CCBs (p > 0.1), it was increased up to 4-fold by AG1478 and PD153035. B, in contrast, NT binding was increased only slightly (<35%) by tyrosine kinase inhibitors, although it was increased up to 3-fold by NIF.

View this table: [in this window] [in a new window]   TABLE 5 Effects of CCBs on PC3 cell binding of ligands specific for bombesin, vasopressin, 2-adrenergic, and EGF receptors

Other Channel Agents Did Not Increase NT Binding. To assess drug specificity, we tested agents toward other ion channels for effects on NT binding. We focused on agents that might alter the movement of Ca²⁺, Na⁺, and K⁺ since NT binding to isolated membranes was known to be inhibited by these metal ions (Carraway et al., 1993). A variety of agents toward other types of channels did not enhance NT binding to PC3 cells (Table 4). These included an N-type Ca²⁺ channel blocker (-conotoxin), Ca²⁺ release inhibitors (ryanodine, dantrolene), K⁺ channel blockers (glibenclamide, diazoxide, tetraethylammonium), an Na⁺ channel blocker (amiloride), and an Na⁺ channel opener (veratridine). These results indicated that the NT binding response displayed a degree of drug specificity.

Enhancement of Cell Binding by CCBs Was Relatively Specific to NT. To assess receptor specificity, we tested CCBs for effects on PC3 cell binding of ligands specific for other GPCRs and for EGFR. Radioreceptor assays were developed for ₂-adrenergic, bombesin, and V_1a-vasopressin receptors as well as for EGFR. Table 1 shows the ligands used and the binding parameters determined. For NT, bombesin, and EGF receptors agonist ligands were used; the others were antagonists. Assessing the effects of CCBs, we found that NIF, phloretin, verapamil, and SKF-96365 did not enhance ₂-adrenergic, V_1a-vasopressin, and EGF receptor binding to PC3 cells (Table 5). However, bombesin receptor binding was elevated slightly (19%) by NIF (Table 5). ₂-adrenergic receptor binding was, in fact, decreased by these agents (Table 5), but this was due to a direct competition with ¹²⁵I-pindolol. This conclusion was based on the structural resemblance of these agents to pindolol and the fact that ¹²⁵I-pindolol binding to PC3 cell membranes was inhibited in a similar manner (for results, see the footnote to Table 5). Cell binding for the vasopressin receptor was also diminished by these drugs (Table 5); however, this could not be attributed to a direct competition with ¹²⁵I-HOLVA (Table 5 footnote). These data indicated that the robust elevation in cell binding was specific to NTR1, although the bombesin receptor responded to a lesser degree.

Inhibition of IP Formation by CCBs Was Relatively Specific to NT. To examine receptor specificity, we tested the ability of NIF to inhibit IP formation in response to GPCR agonists known to stimulate PLC. Preliminary dose-response experiments showed that a maximal dose of NT (30 nM), bombesin (20 nM), and ATP (10 µM) stimulated IP formation by 5-fold, 15-fold, and 17-fold, respectively. When PC3 cells were pretreated with varying amounts of NIF, we found that the response to this dose of NT was inhibited as much as 69%, whereas that for bombesin was inhibited 19%, and that for ATP was not inhibited (Fig. 4A). When the dose of each agonist was varied, we found that the percentage of inhibition by 15 µM NIF was independent of the level of stimulation. Thus, at each dose the response to NT was inhibited 64%, whereas that for bombesin was inhibited 15% and that for ATP was not inhibited (Fig. 4B). These results indicated that the robust inhibition of IP formation by NIF was specific to NT, although the response to bombesin was also inhibited to a lesser degree.

View larger version (21K): [in this window] [in a new window]   Fig. 4. NIF inhibited NT- and bombesin-, but not ATP-induced, IP formation. Agonist-induced IP formation was measured in PC3 cells. A, plots show inhibitory effects of various doses of NIF on the response to a maximal dose of NT (20 nM), bombesin (10 nM), and ATP (10 µM). Results are from three experiments. B, plots show inhibitory effects of 15 µM NIF on responses to various doses of NT (0.2-20 nM), bombesin (0.1-10 nM), and ATP (0.2-10 µM), plotted as a function of fold enhancement of basal IP formation. Results are from three experiments.

CCBs Enhanced Photoaffinity Labeling of NTR1. NTR1 is a 46-kDa protein that has been immunologically characterized (Boudin et al., 1995) and labeled using UV-activatable cross-linkers (Mazella et al., 1988). Initially, we used Western blotting to verify the specificity of our antiserum (Ab-NTR1) raised toward the C terminus of human NTR1. Whereas extracts of rat brain gave a single band at 50 kDa, PC3 cells gave this parent protein, along with a 33-kDa fragment (Fig. 5A), in keeping with published results (Boudin et al., 1995). Next, we used UV light to incorporate ¹²⁵I-(4-azido-Phe⁶)-NT into PC3 cells treated with CCBs or control, and we assessed the incorporation of radioactivity into immunoprecipitated NTR1. The results (Fig. 5B) showed that the radioactivity associated with NTR1 was enhanced by NIF (2.8-fold; p < 0.001), phloretin (1.8-fold; p < 0.05), and verapamil (1.5-fold; p < 0.05) as compared to the control. For each agent the increase in immunoprecipitated radioactivity (Fig. 5B) was similar to the increase in NT binding to PC3 cells seen at the appropriate dose (Fig. 1C). SDS-PAGE and autoradiography on selected samples verified the presence of 50- and 33-kDa radiolabeled proteins (data not shown). These results indicated that CCBs enhanced NT binding by increasing the association of ¹²⁵I-NT with NTR1; however, they did not rule out possible interactions with other NT receptors.

View larger version (21K): [in this window] [in a new window]   Fig. 5. CCBs enhanced photoaffinity labeling of NTR1. A, Western blot, representative of three experiments, verifying the specificity of Ab-NTR1 toward human NTR1. Lane numbering: PC3 cells (lanes 1, 4), rat cerebral cortex (lane 2), rat hypothalamus (lane 3). B, plot showing the effect of CCBs on cross-linking of 125I-(4-azido-Phe6)-NT to immunoprecipitated NTR1. PC3 cells were incubated 10 min in 50 µM NIF, 50 µM phloretin, 100 µM verapamil, or 0.5% DMSO at 37°C. 125I-(4-azido-Phe6)-NT (3 x 105 cpm/ml; 0.15 nM) was added to all dishes and 1 µM NT to some dishes. After 25 min, cells were washed, UV-irradiated, and lysed. Cell membranes were isolated and solubilized NTR1 was immunoprecipitated. After washing, precipitates were counted using a gamma counter and data were expressed relative to control (100%), which typically gave 5000 cpm. The drugs enhanced cross-linking 1.5- to 2.8-fold, and 1 µM NT reduced it by >90%. Results are from four experiments, except NIF (eight experiments).

Cell-Surface Binding versus Internalization. Cell-surface binding of ¹²⁵I-NT was enhanced by NIF to a similar extent when assessed by three different methods (Fig. 6). NIF increased surface binding 2.4-, 2.2-, and 2.7-fold, respectively, as measured at 4°C (Fig. 6A), 37°C in the presence of phenylarsine oxide (Fig. 6A), and 37°C by acid washing (Fig. 6B). Internalization of ¹²⁵I-NT was 68 to 72% of total binding in the presence or absence of NIF (Fig. 6B). In addition, the internalization rate at 37°C for cell-surface ¹²⁵I-NT, previously bound to cells at 4°C in the absence of drugs, was unaffected by 50 µM NIF, 50 µM phloretin, and 50 µM verapamil. Internalization rates (%/min; n = 12 from two experiments) were control, 8.6 ± 0.6; NIF, 8.0 ± 0.6; phloretin, 8.1 ± 0.7; verapamil, 9.2 ± 0.7, which did not differ significantly (p > 0.1). These results indicated that these agents increased cellular NT binding by enhancing the interaction of NT with NTR1 rather than by enhancing the internalization rate for the NT-NTR1 complex.

View larger version (19K): [in this window] [in a new window]   Fig. 6. NIF enhanced cell-surface binding of 125I-NT (A) without altering the percentage of 125I-NT internalized by PC3 cells (B). A, to study the cell-surface component of 125I-NT binding we used method 1, incubation for 2 h at 4°C, and method 2, incubation at 37°C in the presence of 10 µM phenylarsine oxide. NIF enhanced cell-surface binding by 2.3-fold (method 1) and 2.2-fold (method 2). B, to study cell-surface binding and internalization we used method 3, incubation at 37°C followed by acid washing. Internalization of 125I-NT was 71 ± 2% of total binding (control) and 68-72% (NIF). NIF (50 µM) enhanced cell-surface binding (2.8-fold) and internalization (2.6-fold) similarly. Results are from three experiments (A) and four experiments (B).

NTR1 Affinity versus NTR1 Number. CCBs enhanced binding and increased the steepness of the NT displacement curve. When the NT displacement data were expressed as percentage of maximal binding, CCBs shifted the displacement curves to the left by a factor of 2 to 3 (Fig. 7A). In three experiments the K_i value for NT was decreased from 0.95 ± 0.1 nM (control) to 0.36 ± 0.04 nM (50 µM NIF; p < 0.01), 0.40 ± 0.05 nM (50 µM phloretin; p < 0.01), and 0.61 ± 0.06 nM (100 µM verapamil; p < 0.05). Scatchard analyses indicated that NIF increased the affinity of NTR1 for NT without changing the NTR1 number (Fig. 7B). The calculated NTR1 number (158 ± 9 fmol NTR1/mg protein; n = 9) corresponded to 50,000 receptors/cell (Table 1).

View larger version (19K): [in this window] [in a new window]   Fig. 7. Binding displacement curves (A, C) and Scatchard plots (B) for 125I-NT binding to PC3 cells in the presence and absence of CCBs. Binding of 125I-NT to PC3 cells (15.8 cpm/µg of protein) was increased 2.7-fold by 50 µM NIF, 2.6-fold by 50 µM phloretin, and 2.0-fold by 100 µM verapamil. A, plots show displacement of 125I-NT binding by NT, in which binding was expressed as percentage of control. The agents shifted the curves to the left. The IC50 value for NT was 1.2 nM (control), 0.8 nM (verapamil), 0.5 nM (phloretin), and 0.5 nM (NIF). Results are from a typical experiment repeated twice. B, Scatchard plots for typical experiment showing that NIF increased NTR affinity (apparent Ki: control, 0.93 nM; NIF, 0.33 nM) without increasing receptor number (Bmax: control, 23 fmol/well; NIF, 21 fmol/well). C, plots show displacement of 125I-NT binding by SR48692. In the presence of 50 µM NIF, the curve was shifted slightly (but not significantly) to the right. Results are from typical experiment performed four times.

In contrast, the binding displacement curve for the antagonist SR48692 was shifted slightly to the right in the presence of 50 µM NIF (Fig. 7C), although the K_i was not changed significantly (K_i: control, 12 ± 1.0 nM; NIF, 14 ± 0.8; n = 4; p > 0.1). Taken together, these results indicated that CCBs shifted NTR1 toward a state that displayed an increased affinity for the agonist NT and an unchanged affinity for the antagonist SR48692.

NIF Inhibited NT-Induced Ca²⁺ Influx. Since NT stimulated Ca²⁺ influx in Chinese hamster ovary cells transfected with NTR1 (Gailly, 1998), we tested NT for this ability in PC3 cells. NT enhanced the influx of ⁴⁵Ca²⁺ into PC3 cells, giving an EC₅₀ value (1 nM) similar to that for NT-induced IP formation (results not shown). At doses shown to enhance NT binding (Fig. 1C) and to inhibit NT-induced IP formation (Fig. 2B), NIF inhibited the influx of ⁴⁵Ca²⁺ in response to NT (Fig. 8A).

View larger version (20K): [in this window] [in a new window]   Fig. 8. NT-induced 45Ca influx was inhibited by NIF (A), and NT-induced IP formation was Ca2+-dependent (B). A, in experiments not shown, 45Ca2+ influx into PC3 cells was enhanced 30% by NT (EC50 1.2 nM). The log dose-response plot shows that a 10-min pretreatment of cells with varying doses of NIF inhibited the response to 20 nM NT (IC50 12 µM), without much effect on basal 45Ca2+ influx. Results are from three experiments. B, IP formation in PC3 cells in Locke (1 mM Ca2+) was enhanced 4.5-fold by 20 nM NT (shown as 100% response). The response to NT was inhibited by omitting Ca2+ from Locke, by adding to Locke either 1.1 mM EGTA or 50 µM NIF, or 1.1 mM EGTA plus 50 µM NIF. Although basal IP formation was unaffected by NIF it was increased 2-fold by omitting Ca2+ from Locke or by adding EGTA to the Ca2+-containing Locke. Inhibition of the response to NT was not due to a ceiling effect, since IP formation was stimulated 15-fold by 10 nM bombesin and 17-fold by 10 µM ATP in similar experiments. Results are from three experiments.

Ca²⁺-Dependence of NT-Induced IP Formation. Since some PLC isozymes are Ca²⁺-dependent (Rhee and Bae, 1997), the inhibition of NT-induced IP formation by doses of NIF that diminished NT-induced Ca²⁺ influx suggested that Ca²⁺ influx might participate in the stimulation of PLC. Consistent with this, NT-induced IP formation was inhibited by omitting Ca²⁺ from Locke buffer, by adding Ca²⁺ chelator EGTA to Locke, or by adding NIF to Locke (Fig. 8B). Paradoxically, the removal of Ca²⁺ elevated basal IP production 2-fold (see Fig. 8 legend), perhaps by mobilizing internal Ca²⁺ stores. However, inhibition of the NT response was not due to a ceiling effect, since IP production could be elevated 15-20-fold by bombesin and ATP (see Fig. 8 legend).

Ionomycin stimulated IP formation, reproducing as much as 63% of the response to NT. IP formation (percentage of control) was 2 µM ionomycin, 139 ± 6% (p < 0.01); 20 µM ionomycin, 324 ± 14% (p < 0.01); 30 nM NT, 457 ± 12% (p < 0.01); n = 4 experiments. When added 2 min after a maximal dose of NT (30 nM), low doses of ionomycin (2-10 µM) enhanced the response to NT. IP formation (percentage of control) was 10 µM ionomycin, 157 ± 5 (p < 0.01); NT, 366 ± 20 (p < 0.01); NT plus ionomycin, 465 ± 9 (p < 0.001); n = 4 experiments. In contrast, a maximal dose of ionomycin gave less than additive enhancement of the response to NT. IP formation (percentage of control) was 25 µM ionomycin, 322 ± 11 (p < 0.001); NT, 384 ± 14 (p < 0.001); NT plus ionomycin, 476 ± 15 (p < 0.001); n = 4 experiments. These data suggested that the inhibition of NT-induced IP formation by CCBs may have been partly attributable to a change in Ca²⁺ influx.

Ca²⁺-Dependence of NT Binding. In six experiments, removal of Ca²⁺ from the buffer elevated NT binding significantly [NT binding (percentage of control) 2 mM EGTA, 125 ± 5; p < 0.01]. However, relative to the effects of CCBs (Fig. 1), this effect was very small. These data suggested that only a small part (10%) of the effect of Ca²⁺ channel agents on NT binding might be attributed to a change in Ca²⁺ influx.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
This is the first report that CCBs exert major effects on NTR1 binding or even, for that matter, on GPCR binding. Although NT binding to NTR1 was increased dramatically by these agents, NTR1-mediated effects on Ca²⁺ influx and IP formation were inhibited. Drugs representing three major classes of VGCC blockers enhanced NT binding, giving an efficacy order similar to that for peripheral vasodilation (Triggle, 1999). Although the most potent agents, DHPs, were regarded as specific for L-type VGCC (Triggle, 2003), their effects on NT binding and bioactivity occurred in a dose range shown to alter SOCC behavior (Harper et al., 2003). Furthermore, CCBs selective for SOCC elevated NT binding and inhibited NT-induced IP formation. Thus, the effects on NTR1 function were associated predominately with agents having the ability to block SOCC, although SOCC involvement in these actions was not proven.

Enhancement of NT binding by CCBs was drug-specific, receptor-specific, and could not be explained by enhanced tracer stability, membrane partitioning, or metabolic trapping. Under the same conditions that increased NT binding 3-fold and using ¹²⁵I-ligands with similar specific activities, binding for ₂-adrenergic, V_1a-vasopressin, and EGF receptors was not increased, and binding for bombesin receptor was increased <20% by CCBs. Although the results suggested that the effect was specific for NTR1, it was possible that other GPCRs could respond under proper conditions, e.g., agonist ligands might have been necessary for the enhancing effect to manifest itself. The two GPCR binding assays that gave increases in response to CCBs, NTR1 (200% increase) and bombesin (20% increase), used agonist ligands. Although difficult to understand at this time, it is interesting that the V_1a-vasopressin assay, which used an antagonist ligand, gave decreased binding in response to CCBs. Like the increase observed in NT binding, the decrease in vasopressin binding required intact cells (Table 5), and thus was not due to competition at the ligand binding site. Since NTR1, bombesin, and V_1a-vasopressin receptors signal via G_q/11, this suggests that the associated G-protein may be an important determinant of these effects. Although some CCBs decreased ¹²⁵I-pindolol (antagonist) binding to ₂-adrenergic receptors that signal via G_s, this was due to competition at the ligand binding site (Table 5).

That the increase in NT binding involved an enhanced interaction of NT with NTR1 was shown by photoaffinity labeling of immunoprecipitated NTR1. Augmentation of NT binding was not due to an increase in cell-surface receptors or to a change in receptor internalization. The binding of NT has been shown to initiate internalization of the NT-NTR1 complex, a process involving sortilin (Chabry et al., 1993). Stimulation of this process could conceivably lead to an apparent increase in cellular NT binding. However, we found that CCBs did not promote NT internalization and they did not change the apparent number of receptors participating in binding. Instead, the NT-displacement curve was shifted to the left, with an associated decrease in K_i and no change in NTR1 number. Classically, GPCRs display higher affinity for agonists, but not for antagonists, when they are in the coupled state as compared to the uncoupled state. CCBs increased the affinity of NTR1 for agonist NT without altering that for antagonist SR48692. Based on this, we propose that CCBs trap NTR1 in a G-protein-coupled state that exhibits increased affinity for NT. Although NT-induced IP formation was also inhibited, it is not known whether the "high-affinity" state of NTR1 exhibits a reduced ability to activate PLC. However, Paton's rate theory of drug-receptor interaction would predict that increased affinity (associated with a decreased offset rate) would lead to decreased potency (Paton, 1961). Thus, if NTR1 is unable to release NT, it may be less efficacious.

An unexpected outcome was the finding that Ca²⁺ influx participated in the activation of PLC by NT. While other workers have shown that Ca²⁺ was required for PLC action in vitro, agonist-induced IP formation in cells was generally insensitive to removal of extracellular Ca²⁺ (Rhee and Bae, 1997). In contrast, we found 1) that NT-induced IP formation was enhanced by Ca²⁺ ionophore and inhibited by Ca²⁺ removal; 2) that Ca²⁺ ionophore stimulated IP formation, reproducing about half the NT response; and 3) that NT increased Ca²⁺ influx. Since NT can stimulate capacitative Ca²⁺ entry through SOCC (Gailly, 1998), it is likely that SOCCs contribute to the Ca²⁺ component of PLC activation by NT. Consistent with this, we found that the ability to inhibit NT-induced IP formation was associated with SOCC-directed agents. In addition, NIF inhibited NT-induced ⁴⁵Ca²⁺ uptake and NT-induced IP formation over the same dose-range. Unfortunately, we did not test other SOCC blockers for effects on ⁴⁵Ca²⁺ uptake.

Determining the PLC isotype(s) expressed by PC3 cells may be key to understanding these findings. PLCs are classified into three categories (PLC, PLC, and PLC) that exhibit distinct regulatory properties. While PLC is activated by -subunits of G_q/11-type G-proteins and G-subunits from other G-proteins, and PLC is regulated by tyrosine kinases (Rhee and Bae, 1997), PLC is activated by [Ca²⁺] in the physiologic range (Allen et al., 1997). Rhee and Bae (1997) proposed that PLC activation might occur secondary to receptor-mediated activation of PLC via the ensuing elevation in intracellular Ca²⁺. This could provide an explanation for our results, given that Kim et al. (1999) have shown PLC1-activation mediated by the capacitative Ca²⁺ entry following bradykinin-stimulation of PC12 cells. Since PC3 cells express PLC and PLC isoforms (Carraway, unpublished results), it is possible that PLC might be activated by capacitative Ca²⁺ entry following NT-induced stimulation of PLC. Given that removing Ca²⁺ from the buffer inhibited NT-induced IP formation by 70%, this mechanism could account for the majority of IP formed during prolonged NT stimulation. Paradoxically, removing Ca²⁺ from the buffer was by itself a weak stimulus. Basal IP formation increased 2-fold when Ca²⁺ was omitted from or EGTA was added to the Ca²⁺-containing buffer. This effect may have involved the release of Ca²⁺ from internal stores. In preliminary experiments we have shown that thapsigargin, a stimulator of internal Ca²⁺ release, elevates basal IP formation 2-fold.

Enhancement of NT binding by CCBs was always associated with inhibition of NT-induced IP formation. The efficacy order and potencies in these two assays were similar for the agents tested (Table 2). Furthermore, NIF altered bombesin receptor binding and bombesin-induced IP formation precisely as it did for NT, only to a lesser extent. These similar drug and receptor dependencies suggested that these effects came about coordinately or that they were separate events with similar chemical sensitivity. Supporting the latter hypothesis was the different Ca²⁺-dependence of these effects. Whereas NT binding was largely Ca²⁺-independent, NT-induced IP formation was partly Ca²⁺-dependent. Although both effects were associated with SOCC-inhibiting drugs, the rank order of potency (NIF > SKF-96365 > miconazole > trifluoperazine) differed from that for inhibition of SOCC conductance (miconazole > NIF > trifluoperazine > SKF-96365) measured in HL-60 cells (Harper and Daly, 1999, 2003). One possibility is that PC3 and HL-60 cells express different Ca²⁺ channels, e.g., the six mammalian Trp genes can create multiple, functionally diverse Ca²⁺ channels that give complex responses to GPCR agonist activation and store depletion (Zhu et al., 1998). Another possibility is that Ca²⁺ channel occupation per se mediated CCB action, since Ca²⁺ channels interact with GPCRs (Grazzini et al., 1996) and G-proteins (De Waard et al., 1997), and since channel structure and conductance could depend on different drug properties. Another possibility is that CCBs target sites other than Ca²⁺ channels to alter NTR1 structure, and that this action alone enhances binding and obviates NT-induced IP formation. At this time, the simplest explanation is that CCBs produce two effects: 1) they enhance NTR1 binding (and to a lesser extent) bombesin binding; and 2) they inhibit NT-induced (and to a lesser extent) bombesin-induced IP formation. Although changes in Ca²⁺ influx and Ca²⁺ channel interactions might contribute, especially to (2), it seems likely that other targets are also involved. These findings can be summarized as depicted in Fig. 9.

View larger version (17K): [in this window] [in a new window]   Fig. 9. Model depicting the effects of CCBs on NT binding and NT-induced IP formation. 1, by an indirect, Ca2+-independent mechanism, these drugs shift NTR1 into a "high-affinity" state. If the high-affinity state of NTR1 is unable to activate PLC, this would explain the associated inhibition of NT-induced IP formation. 2, alternatively, NT-induced IP formation is inhibited by the blocking of the SOCC, which mediates the Ca2+ entry involved in activation of PLC. 3, another possibility is that these drugs alter some aspect(s) of cellular Ca2+ handling such that influxed Ca2+ is unable to activate PLC.

The effects of CCBs on NTR1 resemble those observed when EGFR is treated with tyrosine kinase inhibitors (Arteaga et al., 1997). Although EGF binding is increased greatly by AG1478 and PD153035 (as shown here), EGFR is unable to autophosphorylate in response to EGF and downstream responses are blocked (Lichtner et al., 2001). Tyrosine kinase inhibitors interact directly with EGFR, and the high-affinity state has been identified as an inactive dimer (Lichtner et al., 2001). CCBs do not interact directly with NTR1 since they do not increase NT binding to isolated cell membranes. However, it might be worthwhile to test the possibility that the phosphorylation state or polymerization state of NTR1 is indirectly altered by CCBs.

The DHPs, nimodipine and NIF, were the most potent (threshold dose, 1 µM) and most efficacious agents tested to elevate NT binding. NIF was also the most effective agent to inhibit NT-induced IP formation. Given that blood levels of DHPs in patients can approach the micromolar range and that DHPs concentrate in membrane fractions (Mason et al., 1992), it is possible that NT binding and bioactivity are altered in humans receiving these drugs. Whether any of the effects of these drugs on cardiovascular function involve NT is not known; however, NT is present throughout the cardiovascular system, where it can produce vasodilation and exert ionotropic and chronotropic effects (Ferris, 1989).

In conclusion, CCBs exert indirect effects in PC3 cells leading to 1) a dramatic increase in cellular NT binding and a smaller increase in bombesin binding; and 2) a dramatic inhibition of NT-induced IP formation and a smaller inhibition of the response to bombesin. Although changes in Ca²⁺ influx and Ca²⁺ channel interactions might contribute, especially to the latter response, it seems likely that other targets are involved.

	Acknowledgements

 
We thank Li Ming Tseng, Amy Wu, and Sheryl Dooley for laboratory help, and Anne Przyborowska for graphics.

	Footnotes

 
This work was supported by Department of Defense (DOD) Grant DAMD17-00-1-0528 and by National Institutes of Health (NIH) Center Grant 5P30-DK32520, although the opinions expressed in the manuscript are not necessarily those of the DOD or the NIH. Part of this material was presented as Abstract P3-576 at the 81st Annual Meeting of the Endocrine Society in June, 1999.

DOI: 10.1124/jpet.102.052688.

ABBREVIATIONS: NT, neurotensin; NTR1, type 1 NT receptor; PLC, phospholipase C; IP, inositol phosphate; CCBs, Ca²⁺ channel blockers; DHP, dihydropyridine; NIF, nifedipine; VGCC, voltage-gated Ca²⁺ channel; SOCC; store-operated calcium channel; HOLVA, des-Gly-[Phaa¹, D-Tyr(Et)²,Lys⁶,Arg⁸]-vasopressin; SKF-96365, 1-[-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenyl]-1H-imidazole; DMSO, dimethylsulfoxide; EGF, epidermal growth factor; HPLC, high-performance liquid chromatography; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PMSF, phenylmethyl sulfonylfluoride; EGFR, EGF receptor; GPCR, G-protein-coupled receptor; SR48692, {2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl)carbonylamino]tricyclo(3.3.1.1 [EC] .^3.7)decan-2-carboxylic acid}; AG-1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline; PD-153035, 4-[(3-bromophenyl)amino]6,7-dimethoxyquinazoline.

¹ This does not necessarily indicate that the association rate constant is increased, since the apparent rate is a function of association, dissociation, internalization and other processes.

Address correspondence to: Dr. Robert E. Carraway, Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. E-mail: robert.carraway{at}umassmed.edu

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