Abstract
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-[β-[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 β2-adrenergic, V1a-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)δ, while the Ca2+-independent part probably involved PLCβ. 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 PLCδ; and 2) they enhance NTR1 affinity by an unexplained Ca2+-independent mechanism.
Footnotes
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↵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.
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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.
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DOI: 10.1124/jpet.102.052688.
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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-[β-[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(3.3.1.1.3.7)decan-2-carboxylic acid}; AG-1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline; PD-153035, 4-[(3-bromophenyl)amino]6,7-dimethoxyquinazoline.
- Received April 4, 2003.
- Accepted July 8, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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