Neurotensin elevates cytosolic calcium in small cell lung cancer cells

Peptides,Vol. 10, pp. 1217-1221. o Pergamon Press plc, 1989. Printed in the U.S.A.

0196-9781/89 $3.00 + .00

Neurotensin Elevates Cytosolic Calcium in Small Cell Lung Cancer Cells J U L I E S T A L E Y , * G A R Y F I S K U M , * T H O M A S P. DAVIS1" A N D T E R R Y W. M O O D Y .1

*Department of Biochemistry, The George Washington University School of Medicine and Health Sciences, Washington, DC 20037 and "pDepartment of Pharmacology, University of Arizona College of Medicine, Tucson, AZ 85724 R e c e i v e d 27 January 1989

STALEY, J., G. FISKUM, T, P. DAVIS AND T. W. MOODY. Neurotensinelevatescytosoliccalcium in small cell lung cancer cells. PEPTIDES 10(6) 1217-1221, 1989.--The ability of neurotensin (NT) to elevate cytosolic Ca2÷ in small cell lung cancer (SCLC) cells was investigated using the fluorescent Ca2+ indicator Fura 2-AM. Using SCLC cell line NCI-H345, NT elevated cytosolic Ca2+ levels in a concentration-dependent manner. Using a 10 nM dose, NT and C-terminal fragments such as NT(8-13) but not N-terminal fragments such as NT(1-8) elevated the cytosolic Ca2÷ levels. Because EGTA (5 mM) did not affect the NT response, NT may cause release of Ca 2+ from intracellular stores. These data indicate that SCLC NT receptors may use Ca2+ as a second messenger. Neurotensin

Small cell lung cancer

Cytosolic calcium

caused by BN was reversed by BN receptor antagonists (5). Here we investigated if NT and NT-related fragments elevate cytosolic Ca 2÷ levels in SCLC cells.

NEUROTENSIN (NT) is a 13 amino acid peptide which may function as a modulatory agent in the CNS (8). It is released from brain neurons (17), binds to CNS receptors (18,35) and is a potent analgesic, hypotensive, hypothermic and hyperglycemic agent after injection into the CNS (6, 12, 28, 30). Besides being present in normal tissues such as the mammalian brain, NT is present in human bronchial biopsy specimens (36), rat medullary thyroid carcinoma (37) and human small cell lung cancer (SCLC) cells (26). Also, SCLC cells, which are enriched in their neuroendocrine properties, have high levels of bombesin (BN)-like peptides (14, 24, 36). NT and BN-like peptides are secreted from SCLC cells into the tissue culture medium (24,26). These peptides may diffuse and bind to NT and BN receptors which are present on the cell surface (1,25). While the role of NT in SCLC remains unknown, BN-like peptides function as autocrine growth factors (13). The second messengers of NT and BN have only recently been studied. In human colonic adenocarcinoma cell line FIT29, nanomolar concentrations of NT stimulate phosphatidylinositol turnover (2). In neuroblastoma clone N I E l l 5 , NT elevates cyclic GMP levels (3) and inhibits cyclic AMP formation (7). While the second messengers utilized by NT in SCLC cells remain unknown, BN stimulates phosphatidylinositol turnover and the resulting inositol 1,4,5-trisphosphate released elevates cytosolic Ca 2÷ levels (16,23). Within 30 seconds after the addition of nanomolar concentrations of BN or the structurally related gastrin releasing peptide, the cytosolic Ca 2÷ levels rose in SCLC cell line NCIH345 from 150 to 190 nM (27). BN caused release of cytosolic Ca z+ from intracellular stores and the increase in cytosolic Ca 2÷

METHOD SCLC cell line NCI-H345 was cultured in SIT medium (RPMI-1640 containing 3 × 10 - 8 M Na2SeO 3, 5 ~g/ml insulin and 10 p,g/ml transferrin) supplemented with 2.5% heat-inactivated fetal calf serum (Biofluids, Rockville, MD). The cells were cultured in a humidified atmosphere of 5% CO2/95% air at 37°(2. New media was added to the cells 24 hours before harvesting. Cells were harvested by centrifugation at 1000 × g for 10 minutes followed by resuspension in SIT medium. The cells were centrifuged an additional time and resuspended in SIT medium containing 10 mM HEPES (pH 7.4). The cells (2.5 x 106/ml) were incubated with 5 p,M Fura 2 AM (Caibiochem, LaJolla, CA) at 37°C for 40 min using a shaking water bath. Unloaded Fura 2 was removed by centrifugation at 150 x g for 3 minutes. Cells were resuspended in SIT/HEPES (2 ml) and transferred to a spectrofluoremeter cuvette containing a magnetic stirrer and maintained at 37°C. Neurotensin and analogues (Peninsula Laboratories, San Carlos, CA) were injected (2-20 Ixl) and the spectra recorded using an excitation wavelength of 340 nm and an emission wavelength of 510 nm. The integrity of the peptides was investigated using HPLC techniques. The peptides were characterized using a Beckman Ultrasphere-ODS (25 x 4.6 ram) column and a Waters Associates Model M6000A HPLC equipped with 2 Model 510 pumps and a

~Rexluests for reprints should be addressed to Dr. Terry W. Moody, Department of Biochemistry, The George Washington University Medical Center, 2300 Eye St. N.W., Washington, DC 20037.

1217

1218

STALEY, FISKUM, DAVIS AND MOODY

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0.1 nM NT

1.0 nM NT

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100 nM NT

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FIG. 1. Dose-responsecurve of NT. Cell line NCI-H345 was loaded with Fura-2 as described in the Method section and the fluorescence was determinedafter the additionof (A) 0.1 nM NT, (B) 1 nM NT, (C) 10 nM NT and (D) 100 nM NT. This experimentis representativeof 3 others. 680 Solvent Programmer. Separations were accomplished using a curvilinear gradient of 21-30% acetonitdle over 40 minutes and the solvent was 0.05 M NaH2PO4 (pH 2.4). UV absorbance was detected at 210 nm and all glassware used, including the WISP autosampler vials, were silanized with 5% dimethyldichlorosilane to minimize peptide loss. The metabolism of NT by intact SCLC cells was also determined. SCLC cells were harvested at 1250 × g for 10 min, then resuspended in sterile RPMI-1640 containing no serum or additives to a cell concentration of 2 × 10 6 cells/ml. Cells (0.4 × l 0 6) were incubated at 37 _+0.1°(2 with 100 0.M NT; total volume was 200 ~l. After time course incubation (5-90 min), the enzymatic activity was halted by the addition of trichloroacetic acid (25 Ixl), the samples centrifuged and the supematant assayed by HPLC. RESULTS Previously, we showed that BN-like peptides but not VIP or SRIF elevated the cytosolic Ca2+ levels in SCLC cells (27). In the present study, NT elevated the cytosolic Ca2÷ levels in a concentration-dependent manner. Low levels of NT (0.1 riM) increased the cytosolic Ca2+levels and a steady state value was reached after approximately 30 seconds (Fig. 1); the cytosolic Ca2+ levels returned to basal values approximately 6 minutes after NT addition. Using 1 nM NT, the amplitude of the response was greater. The amplitude of the response was maximal using 10 nM NT, however, the subsequent decline was rapid. Ten nanomolar NT routinely increased the cytosolic Ca2+ levels from 150 +- 10 nM to 270-+ 20 nM using cell line NCI-H345. Using 100 nM NT, the cytosolic Ca2+ levels rapidly increased to a steady state value, then rapidly decreased (Fig. 2). When 5 mM EGTA was added there was a decline in the steady state value of the Fura 2 fluorescence. Nonetheless, when NT was subsequently added there was a rapid increase in the cytosolic Ca2+ levels. These data suggest that NT causes release of Ca2+ from intracellular stores. The structure-activity relationship of NT to elevate cytosolic Ca2+levels was determined. NT, [GIn4]NT and C-terminal fragments such as NT(8-13) and Ac-NT(8-13) increased the cytosolic Ca2+ levels (Table 1). In contrast, N-terminal fragments such as NT(1-8) had no effect on the cytosolic Ca2+ levels. Also, NT(9-13) and [Trpll]NT (10 riM) increased the cytosolic Ca2+ levels. Those analogues enriched in D-amino acids such as [D-TrpH]NT, [D-TyrH]NT, [D-PheH]NT, [D-Arg12]NT and [DLeuI2]NT did not alter the cytosotic Ca2+ levels. We investigated if the compounds which did not alter the cytosolic Ca2+ levels were inactive. [D-PheH]NT (10 nM) had no

100 M NT

t-,-2 Time

FIG. 2, Effect of EGTA on NT response, The cytosolic Ca 2+ levels were determined after the addition of (A) 100 nM NT and (B) 5 mM EGTA followed by the additionof 100 nM NT. This experimentis representative of 2 others.

effect on the cytosolic Ca2"- levels, however, subsequent addition of 10 nM NT increased the cytosolic Ca2+ levels (Fig. 3A). These data suggest that [D-Phel~]NT is inactive at the concentration used. Similar results were obtained for [D-Trp 11]NT, [D-Tyr11]NT, NT(1-8), [D-ArgX2]NTand [D-Leu12]NT (data not shown). When 10 nM NT(8-13) was added there was a transient increase in the cytosolic Ca2+ levels followed by a slow decline (Fig. 3B). Subsequent addition of 10 nM NT caused no increase in the cytosolic Ca2÷ levels. These data suggest that NT(8-13) and NT elevate cytosolic Ca2÷ levels through the same mechanism. NT (10 nM) caused transient increase in the cytosolic Ca2÷ followed by rapid decline which may result from NT receptor down regulation and/or desensitization. Subsequent addition of 1000 nM TABLE 1 A BI L I T Y OF N T A N A L O G U E S T O E L E V A T E CY T O SO L I C Ca 2+

Peptide

Ca2÷ Response

NT NT(8-13) NT(1-8) [Gln']~r Ac-NT(8-13) NT(9-13) ~l'rp"]NT [D.TrplI]NT [D-Tyr11]NT

+ + + + + +

[D-Phel1]NT [D.ArgI2]NT [D.Leu~2]NT The abilityof biT analogues(10 riM) to elevate cytosolicCa2+ levels in SCLC cell line NCI-H345 was determined;positiveresponse (+), inactive (-).

NEUROTENSIN AND CYTOSOLIC Ca2+

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FIG. 3. Effect of NT, NT analogues and BN on the cytosolic Ca2÷ levels. The cytosolic Ca2÷ levels were determined after addition of (A) 10 nM [D-PheH]NTfollowed by 10 nM NT, (B) 10 nM NT(8-13) followed by 10 nM NT and (C) 10 nM NT followed by 1000 nM BN. This experimentis representativeof 2 others.

BN, which causes release of Ca 2÷ from intracellular pools, still caused a robust Ca2÷ response (Fig. 3C). NT and its fragments were fractionated using reversed phase HPLC. NT(I-8), NT(1-11), NT(8-13), NT(9-13), Ac-NT(8-13) and NT eluted at retention times 8, 18.5, 20.5, 23, 26, 29 and 40 min after HPLC injection, respectively (Fig. 4). Because only one major peak of absorbance was detected for each synthetic peptide standard and amino acid values after hydrolysis confirmed content at 98%, the peptides were considered greater than 95% pure. The stability of NT was determined after time course exposure to the SCLC cells. NT was not appreciably metabolized after 5 min. With increasing time, however, NT metabolites were observed. Figure 4 shows the NT metabolites after a 90-minute exposure to

SCLC cells. There was an injection spike 1-3 min after sample injection which likely contains several hydrophilic components. The major peptide peak coeluted with synthetic NT. Also, intermediate peaks of absorbance eluted 13 and 26 min after injection into the HPLC; the former peak is unidentified but the latter peak coeluted at the same retention time as did synthetic NT(8-13). Minor peaks of absorbance eluted at the retention times as did NT(I-11), NT(9-13) and Ac-NT(9-13). NT (8-13) was the major fragment formed during the incubation of NT with NCIH345 (Fig. 4). Because the half-life for NT was calculated to be 84 min, it is not appreciably degraded during the 5 minutes required to perform the cytosolic Ca2÷ assay. DISCUSSION

go

30

FIG. 4. Metabolismof NT by small cell lung cancer cells. NT (100 p.M) was incubatedwith SCLC ceils at 37°C for 90 minutesas described in the Method section and fractionated using HPLC techniques. The figure represents 7 t~g of protein and the elutionpositionof syntheticstandards is indicated.

Previous data indicate that NT may utilize the phosphatidylinositol second messenger system. In HT29 cells, 20-30 seconds after the addition of 100 nM NT, a 275% and 420% increase in inositol trisphosphate and inositol bisphosphate, respectively, occurred (2). Similarly, NT stimulated phosphatidylinositol turnover in N1E115 cells (33) and in brain slices (15). Because inositol trisphosphate causes release of Ca2÷ from intracellular pools, we investigated if NT elevates cytosolic Ca2÷ levels in SCLC cells. NT elevated cytosolic Ca2÷ levels in a dose-dependent manner. A significant Ca2÷ response was observed using only 0.1 nM NT. The amplitude of the response slightly increased and the duration of the response slightly decreased using 1 nM NT. Recent studies indicate that ~25I-[Tyr3]-NTbinds with high and moderate affinity to 2 classes of sites (Kd =0.2 and 5 nM) using rat, guinea pig and human synaptic membranes (31,32). Similarly, 125I[Tyr3]-NT binds to 2 classes of sites (Kd=0.3 and 6 nM) using SCLC cell line NCI-H345 (A. Alien et al., unpublished). These data suggest that occupation of the high affinity NT receptor results in increased cytosolic Ca2+. High concentrations of NT (100 nM) caused a transient increase in the cytosolic Ca2÷ levels followed by a rapid decline. The decline was not due to depletion of intracellular Ca 2+ pools, as after addition of NT, BN, which also releases Ca2+ from intracellular pools, still caused a significant Ca2÷ response. Therefore, NT may cause desensitization and/or down regulation of NT receptors. This was verified by an additional experiment. When SCLC cells, which were loaded with Fura 2, were exposed to 10 nM NT, then centrifuged to remove free NT, and after 30 min reexposed to 10 nM NT, the cytosolic Ca 2÷ response was virtually absent.

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STALEY, FISKUM, DAVIS AND MOODY

NT caused release of Ca 2÷ from intraceUular pools using SCLC cells. Because the NT response was almost identical when EGTA was added to the medium, NT does not appear to open plasma membrane Ca 2+ channels allowing extracellular Ca 2÷ into SCLC cells, but rather causes release of Ca 2÷ from intracellular organelles such as the endoplasmic reticulum. In contrast, NT increased Na ÷ and Ca 2÷ membrane conductance in guinea pig taenia coli smooth muscle resulting in smooth muscle depolarization and contraction (19,20). The structure-activity relationship of various NT analogues was investigated. NT and the C-terminal fragment NT(8-13) increased the cytosolic Ca 2÷ levels. Previously, we found that NT and NT(8-13) strongly inhibited ~25I-NT binding to SCLC cell line NCI-H209 with IC5o values of 4 and 10 nM, respectively (1). Similarly, [Glna]NT and Ac-NT(8-13) increased the cytosolic Ca 2÷ levels. Previously, nM concentrations of NT were required to inhibit binding of radiolabeled NT to rat brain membranes and Ac-NT(8-13), as well as NT(8-13) were slightly more and less potent, respectively, than was NT (21,22). NT(9-13)and [Trp ~~]NT were agonists and increased the SCLC cytosolic Ca 2+ concentrations. Similarly, NT(9-13) and [Trp~ lINT moderately and strongly inhibited binding of radiolabeled NT to rat brain membranes (22,29). The N-terminal fragment NT(1-8) and the NT analogues [D-TrpI1]NT, [D-TyrH]NT, [D-PheI1]NT, [DArg~2]NT and [D-Leu12]NT did not alter the cytosolic SCLC Ca 2÷ levels and were inactive. Previously, we demonstrated that NT(18), [D-Phell]NT, [D-TyrH]NT, [D-Argl2]NT and [D-LeuI2]NT did not inhibit specific ~25I-NT binding to SCLC cells (IC5o> 1000

nM) (1). Similarly, [D-Phe t lINT and [D-Trp I I]NT were less than 1% as potent as NT in inhibiting radiolabeled NT binding to rat brain membranes (21,22). Surprisingly, [D-TyrH]NT and [DPhe ~m]NT are more potent at inducing hypothermia in rats than is NT, because of decreased proteolytic degradation in vivo (10). The synthetic peptides were not appreciably degraded during the 5-minute time course of the cytosolic Ca 2- assay. If NT was incubated with the SCLC cells for 90 minutes, however, substantial degradation of NT occurred to NT(8-13). Previously, a metalloendopeptidase was identified which cleaves NT at the ArgS-Arg9 peptidyl bond (9), an endopeptidase which hydrolyzes the Pro~°-Tyr II (11) bond and endopeptidase 24.11 which hydrolyzes the Tyr 1l-Ile 12 bond (9,11). [D-Tyr 1lINT and [D-Phe I lINT are resistant to degradation by these proteolytic enzymes (10). Recently, Bepler (4) determined that 1 txM NT had no effect on the growth of SCLC. This concentration of NT, however, causes a robust Ca 2÷ response. Thus transient elevation of the cytosolic Ca 2÷ concentration alone may not be sufficient to cause a proliferative response in SCLC cells. In summary, NT and its C-terminal but not N-terminal analogues elevate the cytosolic Ca 2+ concentrations in SCLC cells. Because NT is biologically active on SCLC cells, it may function as a regulatory peptide in this disease. ACKNOWLEDGEMENTS The authors thank Dr. S. St.-Pierre for supplying some of the NT analogues. This research was supported by NCI grants CA33767 and CA42306 to T.W.M. and CA44869 to T.P.D.

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NEUROTENSIN AND CYTOSOLIC Ca 2+

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29. 30.

logical activity and binding properties to rat brain synaptic membranes. J. Biol. Chem. 258:3476-3481; 1983. Mendoza, S. A.; Schneider, J. A.; Lopez-Rivas, A.; Sinnet-Smith, J. W.; Rozengurt, E. Early events elicited by bombesin and structurally related peptides in quiescent swiss 3T3 cells. II. Changes in Na ÷ and Ca2+ fluxes, Na +, K ÷ pump activity, and intra-cellular pH. J. Cell Biol. 102:2223-2233; 1986. Moody, T. W.; Pert, C. B.; Gazdar, A. F.; Carney, D. N.; Minna, J. D. High levels of intracellular bombesin characterize human small cell lung cancer. Science 214:1246-1248; 1981. Moody, T. W.; Carney, D. N.; Cuttitta, F.; Quattrocchi, K.; Minna, J. D. High affinity receptors for bombesin/GRP-like peptides on human small cell lung cancer. Life Sci. 36:105-113; 1985. Moody, T. W.; Carney, D. N.; Korman, L. Y.; Gazdar, A. F.; Minna, J. D. Neurotensin is produced by and secreted from classic small cell lung cancer cells. Life Sci, 36:1727-1732; 1985. Moody, T. W.; Murphy, A.; Mahmoud, S.; Fiskum, G. Bombesinlike peptides elevate cytosolic calcium in small cell lung cancer cells. Biochem. Biophys. Res. Commun. 147:189-195; 1987. Nemeroff, C. B.; Hernandez,C.; Luttinger, D.; Kalivas, P. W.; Prange, A. J., Jr. Alterations in nociception and body temperature after intracisternal administration of neurotensin, 13-endorphin and other endogenous peptides and morphine. Proc. Natl. Acad. Sci. USA 76:5368-5371; 1979. Quirion, R.; Gaudreau,P.; St-Pierre, S.; Rioux, F.; Pert, C. Autoradiographic distribution of 3H-neurotensin receptors in rat brain: Visualization by tritium-sensitive film. Peptides 3:757-763; 1982. Rioux, F.; Quirion, R.; St.-Pierre, S.; Regoli, D.; Jolicoeur, F.; Belanger, F.; Barbeau, A. The hypotensive effect of central admin-

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istered neurotensin in rats. Eur. J. Pharmacol. 69:241-247; 1980. 31. Sadoul, J. L.; Kitabgi, P.; Rostene, W.; Javoy-Agid, F.; Agid, Y.; Vincent, J.-P. Characterization and visualization of neurotensin binding to receptor sites in human brain. Biochem. Biopbys. Res. Commun. 120:206-213; 1984. 32. Sadoul, J. L.; Mazella, J.; Amar, S.; Kitabgi, P.; Vincent, J.-P. Preparation of neurotensin selectively iodinated on the tyrosine 3 residue. Biological activity and binding properties on mammalian neurotensin receptors. Biochem. Biophys. Res. Commun. 120:812819; 1984. 33. Snider, R. M.; Kyes, S. A.; Seguin, E. B.; Agaroff, B. W. Inositol lipid labeling produced by muscarine, histamine H~ and thrombinreceptor stimulation in neuroblastoma cells. Soc. Neurosci. Abstr. 10:276; 1984. 34. Takuwa, N.; Takuwa, Y.; Bollag, W. E.; Rasmussen, H. The effects of bombesin on polyphosphoinositide and calcium metabolism in Swiss 3T3 cells. J. Biol. Chem. 262:182-188; 1987. 35. Uhl, G. R.; Kuhar, M. J.; Snyder, S. H. Neurotensin, a central nervous system peptide: Apparent receptor binding in brain membranes. Brain Res. 130:299-313; 1977. 36. Wood, S. M.; Wood, J. R.; Ghatei, M. A.; Lee, Y. C.; O'Shaughnessy, D.; Bloom, S. R. Bombesin, somatostatin and neurotensin-like immunoreactivity in bronchial carcinoma. J. Clin. Endocrinol. Metab. 53:1310-1312; 1981. 37. Zeytinoglu, F. N.; Gabel, R. F.; Tashjian, A. H.; Hammer, R. A.; Leeman, S. E. Characterization of neurotensin production by a line of rat medullary thyroid carcinoma cells. Proc. Natl. Acad. Sci. USA 77:3741-3745; 1980.