Peptides, Vol. 12, pp. 1315-1319. ©Pergamon Press plc, 1991. Printed in the U.S.A.
0196-9781/91 $3.00 + .00
Vasopressin Elevates Cytosolic Calcium in Small Cell Lung Cancer Cells I M. H O N G A N D T. W. M O O D Y 2
Department of Biochemistry and Molecular Biology, The George Washington University School of Medicine and Health Sciences, Washington, DC 20037 R e c e i v e d 28 M a r c h 1991 HONG, M. AND T. W. MOODY. Vasopressin elevates cytosolic calcium in small cell lung cancer cells. PEPTIDES 12(6) 1315-1319, 1991.--The ability of vasopressin to elevate cytosolic Ca2÷ in small cell lung cancer (SCLC) cells was investigated. Ten nanomolar vasopressin elevated the cytosolic Ca2÷ in 6 of 8 SCLC cell lines that were loaded with Fura-2 AM. Using SCLC cell line NCI-H345, the effect of vasopressin was dose dependent, being maximal at 100 nM, where the cytosolic Ca2÷ was elevated from 150 to 210 nM. Because addition of 1 mM EGTA had no effect on the vasopressin response, vasopressin released Ca2÷ from intracellular pools. Also, oxytocin weakly elevated the cytosolic Ca2+ . The response to vasopressin was strongly blocked by [(13-mercapto-13,13-cyclopentamethylene propionic acid)l,O-MeTyr2,ArgS]vasopressin and weakly blocked by [(13-mercapto-13,13-cyclopentamethylene propionic acid)l,O-MeTyr2,OmS]vasotocin. These data suggest that V l vasopressin receptors are present on SCLC cells. Small cell lung cancer
Vasopressin
Calcium
Fura-2
VASOPRESSIN (AVP) and oxytocin, which are nonapeptides, are synthesized in the paraventricular and supraoptic nuclei of the hypothalamus in the form of high molecular weight precursor proteins (6). After posttranslational processing, neurophysins and the 9 amino acid AVP and oxytocin result, which have 7 of the same 9 amino acid residues (17). These peptides function as posterior pituitary hormones and AVP regulates osmolarity, blood pressure and volume, whereas oxytocin induces uterine contractions and stimulates milk release from the mammary gland (8). Vasopressin is also active in the brain, where treatment of neonatal and adult animals affects learning and memory, respectively (23,24). After direct injection into the rat brain, AVP and [Cyt6]AVP(5-9) facilitate avoidance behavior (10,11). These actions may be mediated by AVP receptors, which have been detected in the CNS and periphery. The V 2 receptor, which is present in the kidney, stimulates adenylate cyclase activity (7). In contrast, the Via receptor, which is present in the liver, vascular smooth muscle, adrenals, testis and brain, increases phosphatidylinositol (PI) turnover (2, 3, 7, 14, 19). In the liver and hepatocyte, phospholipase C may be coupled to the V l receptor, resulting in inositol-l,4,5-trisphosphate (IP3) and increases in intracellular Ca 2+ . Hypophysial Via receptors also stimulate the PI pathway, resulting in elevated intracellular Ca 2 ÷ (9). The V~b receptor in the anterior pituitary is coupled to adenylate cyclase (9). Previously, immunoreactive AVP and AVP mRNA were reported to be present in some SCLC cell lines (18). In addition, AVP stimulates the clonal growth of certain SCLC cell lines (16). Here we investigated if AVP increased the cytoslic Ca 2÷
in Fura-2 AM-loaded SCLC cells. Structure-activity studies using AVP analogues suggest that V~ receptors are present in SCLC cell line NCI-H345. METHOD The human tumor cell lines were cultured in serum-supplemented medium (RPMI 1640 containing 10% heat-inactivated fetal calf serum) in a humidified atmosphere of 5% CO 2 and 95% air at 37°C. On the day of the assay, cells were harvested by centrifugation at 1000 x g for 10 minutes. Cells were washed and resuspended in assay buffer (SIT medium-RPMI 1640 conmining 3 x 10 - 8 M Na2SeO 3, 5 ixg/ml bovine insulin, and 10 p,g/ml human transferrin). Here we investigated if AVP analogues altered the intracellular Ca 2÷ . For the cytosolic Ca 2 + assays, cell line NCI-H345 was harvested 24 hours after a medium change, washed, and resuspended in SIT media containing 20 mM HEPES/NaOH (pH 7.4). SCLC cells ( 2 . 5 x 106/ml) were incubated with 5 IxM Fura-2 AM (Calbiochem Inc., La Jolla, CA) at 370(2 for 30 minutes in a shaking water bath. Cells were centrifuged at 1 5 0 × g for 2 minutes, and the pellet containing SCLC cells loaded with Fura-2 were resuspended to the same concentration. Fluorescence was monitored via a spectrofluorometer at an excitation and emission wavelength of 340 nm and 510 nm, respectively. The spectrofluorometer was equipped with a temperatureregulated cuvette holder in addition to a magnetic stirrer. Cytosolic Ca 2 + was measured upon addition of various AVP analogs and antagonists.
IPortions of this article were presented at the 12th Annual Winter Neuropeptide Conference, Breckenridge, CO (February, 1991). 2Requests for reprints should be addressed to Dr. Terry W. Moody, GWU Biochemistry Department, 2300 Eye St. N.W., Washington, DC 20037.
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HONG AND MOODY
TABLE 1 EFFECT OF PEPTIDES OF C a 2+ LEVELS IN SCLC CELLS Cell Line
AVP
BN
CCK
NT
NCI-H146 NCI-H187 NCI-H209 NCI-H345 NCI-N417 NCI-H510 NCI-H1092 NCI-H 1694
-+ ++ ++ ++ ++ + .
-+ ++ ++ -++ +
++ + ++ ++ -++ +
-++ ++ ++ --+
.
.
.
Peptides were added at a 10 nM concentration and the Ca2+ response determined in the indicated SCLC cell line. Strong response, + +; weak response, +; no response, --.
RESULTS Previously, we found that neurotensin (NT), bombesin/gastrin releasing peptide (BN/GRP) and cholecystokinin (CCK) elevated cytosolic Ca 2÷ in SCLC cell line NCI-H345 via distinct neuropeptide receptors (15, 20, 21). Table 1 shows that AVP caused a strong Ca 2 ÷ response in 4 of 8 SCLC cell lines examined, including NCI-H209, H345, N417 and H510. In contrast, 10 nM AVP caused a weak response in 2 other cell lines (NCIH187 and H1092), whereas there was no response in 2 other cell lines (NCI-H146 and H1694). Also, CCK, BN and NT caused a Ca 2 ÷ response in 6, 5 and 4 cell lines, respectively, of the 8 cell lines examined. Subsequently, the effect of AVP was investigated using cell line NCI-H345, which had a strong Ca 2 ÷ response to AVP, BN, CCK and NT. Figure 1 shows that AVP elevated the cytosolic Ca 2÷ in a dose-dependent manner. The cytosolic Ca 2 ÷ was minimally affected by 0.1 or 1 nM AVP, whereas 10 nM AVP increased the cytosolic Ca 2÷ from 150 to 190 nM. At 10 nM AVP, the cytosolic Ca 2÷ increased slowly, reaching maximal values after 30 s. Then after a transient steady state, the cytosolic Ca 2 ÷ slowly returned to basal levels within 5 min. At a 100 or 1000 nM
A
0.1 nM AVP
B
1 nM AVP
AVP concentration, the cytosolic Ca 2+ increased from 150 to 210 nM. AVP released Ca 2+ from intracellular organelles. Figure 2 shows that when 10 nM AVP was added to cell line NCI-H345, which was loaded with Fura-2 AM, there was an increase in the fluorescence intensity. When 1 mM EGTA was added to the extracellular medium, there was a sharp reduction in the fluorescence intensity due to extracellular Fura-2 AM going from the Ca 2+ bound to Ca 2÷ free state. When 10 nM AVP was subsequently added, there was still an increase in the fluorescence intensity almost identical to that observed previously. These data suggest that AVP does not open extracellular Ca 2+ channels. Oxytocin, which is structurally similar to AVP, had no effect on the cytosolic Ca 2+ at a 10 nM dose (data not shown). At 100 nM oxytocin, the cytosolic Ca 2+ increased from 150 to 180 nM. At a 1000 nM or 10,000 nM concentration, oxytocin maximally increased the cytosolic Ca 2+ . These data indicate that oxytocin is over 1 order of magnitude weaker at elevating the cytosolic Ca 2 + than is AVP. Oxytocin may elevate cytosolic Ca 2÷ by interacting with AVP receptors. Figure 3B shows that when 10 p,M oxytocin was added, a strong Ca 2+ response was obtained. If 10 nM AVP was subsequently added, the Ca 2+ response was absent. These data indicate that oxytocin and AVP elevate cytosolic Ca 2+ through a similar mechanism. Similarly, if 10 nM AVP was added there was a strong Ca 2+ response (Fig. 3A). If 10 nM AVP was subsequently added, the response was absent, possibly due to receptor desensitization. In contrast, BN, CCK or NT elevated the cytosolic Ca 2 ÷ but had no effect on the AVP Ca 2 + response, whereas pressinoic acid was inactive (data not shown). [D-Argl,D-Pro2,D-Trp7'9,Leull]substance P ([APTTL]SP) was an antagonist in that, if 10 IxM [APTTL]SP was added, there was no Ca 2÷ response (Fig. 3C). If 10 nM AVP was subsequently added, there was no increase in the cytosolic Ca 2 ÷. In addition to [APTTL]SP, [(13-mercapto-13,13-cyclopentam e t h y l e n e propionic a c i d ) l , O - M e T y r 2 , o r n i t h i n e a ] v a s o t o c i n [d(CH2)5,Tyr(Me)2]OVT functioned as a weak antagonist. [d(CH2)5,Tyr(Me)2]OVT (1000 nM) had no effect on the cytosolic Ca 2÷' but decreased the increase in cytosolic Ca 2+ caused by 1 nM AVP (Fig. 4). If 10 nM AVP was subsequently added, there was a slight increase in the cytosolic Ca 2÷ from 150 to 160 nM. If 1000 nM AVP was subsequently added,
C
D
10 nM AVP
100 nM AVP
E
1000 nM AVP
.Z.__
20C
15o
~--- 4 rain-~
~--- 4 rain--~
~--- 4 min-~
~- 4 rain--~
4 rain - - ~
Time, min
FIG. 1. Effect of vasopressin (AVP) on cytosolic Ca2+ . SCLC cell line NCI-H345 was loaded with Fura-2 AM and the ability of (A) 0.1 nM AVP, (B) 1 nM AVP, (C) 10 nM AVP, (D) 100 nM AVP and (E) 1000 nM AVP to elevate cytosolic Ca2+ determined. This experiment is representative of 3 others.
VASOPRESSIN AND SCLC
1317
10 nM VP
E
~
o
=;
4 min
c o
10 nM VP
u.
~9---- 4 rain ----~ Time, min
FIG. 2. Effect of EGTA on AVP response. The ability of 10 nM AVP to elevate the cytosolic Ca 2÷ was determined prior to and after the addition of 1 mM EGTA to the extracellular medium.
there was a strong C a 2+ response. These data indicate that [d(CH2)5,Tyr(Me)2]OVT is a reversible AVP antagonist. [(13-Mercapto-13,13-cyclopenthamethylene propionic acid)l,O MeTyr2]AVP [d(CH2)5,T~yr(Me)2]AVP was a strong AVP antagonist. [d(CH2)5,Tyr(Me)~]AVP (1 nM) had no effect on the cytosolic Ca 2+ , but decreased the amplitude of the Ca 2+ response to 10 nM AVP (Fig. 5A, B). [d(CH2)5,Tyr(Me)2]AVP (100 nM) had no effect on the cytosolic Ca 2÷ , but totally antagonized the response to 10 nM AVP (Fig. 5C). Similarly, 10 nM [d(CH2)5,Tyr(Me)2]AVP blocked the response to 10 nM AVP, whereas 10 nM [d(CH2)5,Tyr(Me)2]OVT had no effect on the response to 10 nM AVP (data not shown). These data indi-
cate that [d(CH2)5,Tyr(Me)2]AVP is approximately 2 orders of magnitude more potent than is [d(CH2)5,Tyr(Me)2]OVT as a AVP antagonist. DISCUSSION
Previously, 100 nM AVP was reported to elevate the cytosolic Ca 2+ in SCLC cell line NCI-H345 and H510, but not large cell cancer cell line NCI-H460 or breast cancer cell lines ZR-75 or MCF-7 (5). Further, the Ca 2+ response to AVP was distinct from that of BN, NT, CCK or bradykinin. Here the pharmacology of the AVP Ca 2 ÷ response was investigated.
C
10 nM AVP
200
150
10 nM AVP
,l
l
f-...__ L I-
10 uM Oxy 10 nMAVP 10 uM (APTTL)SP 10 nM AVP
4 rain - ~
!
~1---- 4 min - - ~
~1--- 4 min
~t -[
Time, min FIG. 3. Pharmacologic specificity of the AVP response. The cytosolic Ca 2 + levels were determined after the addition of (A) 10 nM AVP followed by 10 nM AVP, 03) 10 p,M oxytocin (Oxy) followed by 10 nM AVP and (C) 10 p,M [ A F I ' ~ ] S P followed by 10 nM AVP.
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HONG AND MOODY
1 uM
1 nM
(d(CH2)5,
AVP
]Tyr(Me)2)OVT
10 nM AVP
1000 nM AVP
i
,l 200 4-
150
I_
lO mi.
I-
_1
-I
Time, rain
FIG. 4. Reversibility of an AVP antagonist. [d(CH2)5,Tyr(Me)2]OVT (1 ~M) had no effect on the cytosolic Ca2÷ . AVP was subsequently added at a 1 nM concentration, followed by 10 nM AVP and 1000 nM AVP.
Ca 2+ response caused by 10 nM AVP. These data suggest that the AVP, GRP and CCK-B receptors present on SCLC cell line NCI-H345 are distinct. AVP may elevate the cytosolic Ca 2+ on SCLC cell line NCI-H345 by binding to Via receptors. The EDso for AVP and oxytocin to elevate the cytosolic Ca 2÷ is approximately 5 and 200 nM, respectively. Further, the EDso for [d(CH2)5,Tyr (Me)2]AVP and [d(CH2)5,Tyr(Me)2]OVT to inhibit the increase in cytosolic Ca 2+ caused by 10 nM AVP is approximately 1 and 1000 nM, respectively. Previously, [d(CH2)5,Tyr(Me)2]AVP and [d(CH2)5,Tyr(Me)2]OVT were found to be V] and V 2 receptor antagonists (1,9). For V 1 receptors in the rat septum, the K i values for vasopressin, oxytocin and [d(CH2)5,Tyr(Me)z]AVP to inhibit 3H-AVP binding are 2, 185 and 1 nM, respectively. Previously, it was found that [APTTL]SP, a SP antagonist, also antagonizes SCLC BN/GRP receptors. Thus 10 p,M [APTTL]SP inhibited ~2~I-GRP binding to NCI-H345, the cytosolic Ca 2 ÷ induced by 10 nM BN and clonal growth (4). Also, 10 txM [APTTL]SP inhibited 125I-GRP and 3H-AVP binding to
Ten nM AVP increased the cytosolic Ca 2 + in 6 of 8 SCLC cell lines examined. These data suggest that many, but not all, SCLC cells have functional AVP receptors. Cell lines NCI-H209 and NCI-H345 strongly responded to AVP as well as BN, CCK and NT. Using cell line NCI-H345, however, the mechanisms by which AVP, BN, CCK and NT elevated cytosolic Ca 2÷ were different. If 10 nM AVP was added, there initially was an increase in the cytosolic Ca 2+ from 150 to 190 nM within 30 s, followed by a slow decrease to baseline over the next 5 min. If 10 nM AVP or 10 IxM oxytocin was subsequently added, there was a lack of Ca 2 ÷ response. These data suggest that AVP or oxytocin increase the cytosolic Ca 2 ÷ through a similar mechanism. AVP and oxytocin do not deplete the cell of Ca 2+ because addition of 10 nM, BN, NT or CCK after the addition of 10 nM AVP caused a strong increase in the cytosolic Ca 2÷ . Therefore, AVP and oxytocin may bind to the same receptor. Further, the cytosolic Ca 2÷ response to BN is blocked by [Psila'14,Leul4]BN (13) and the response to CCK blocked by L-365,718 (22), and neither of these compounds inhibits the
A
B
C
I nM (d(CH2)5, 10 nM AVP 20O
Tyr(Me}'2)AVP
100 nM (d(CH2]5, 10 nM AVP
Tyr(Me)2)AVP 10 nM AVP
1
r2
Time, mtn
FIG. 5. Dose response of a AVP antagonist. The cytosolic C a 2 + response was determined after the addition of (A) 10 nM AVP, (B) 1 nM [d(CH2)~,Tyr(Me)2]AVP followed by 10 nM AVP and (C) 100 nM [d(CH2)5,Tyr(Me)2]AVP followed by 10 nM AVP.
VASOPRESSIN AND SCLC
1319
Swiss 3T3 cells, the increase in cytosolic Ca 2 + caused by AVP and BN as well as the increase in 3H-thymidine uptake induced by AVP or BN (25). Because the SP antagonist bears little sequence homology to AVP or BN, it may interact with guanine nucleotide binding proteins impairing the receptor conformation. It remains to be determined if AVP is an autocrine growth factor for SCLC cells. Because the peptide is synthesized and translated in SCLC cells, and is biologically active, it could be an autocrine growth factor. Besides elevating cytosolic Ca 2+ , AVP (10 ng/ml) stimulated the growth of SCLC cells
(16). It remains to be determined if AVP antagonists such as [d(CH2)5,Tyr(Me)2]AVP inhibit the growth of SCLC. In summary, AVP elevates cytosolic Ca 2+ in some SCLC cell lines. Structure-activity studies suggest that V 1 receptors may be present on SCLC. AC~O~E~E~S The authors thank J. Staley for technical assistance and Dr. G. Fiskum for helpful discussions. Supported in part by NCI grant CA-53477.
REFERENCES 1. Bankowitz, K.; Manning, M.; Seto, J.; Haldar, J.; Sawyer, W. H. Design and synthesis of potent in vivo antagonists of oxytocin. Int. J. Pept. Protein Res. 16:382-391; 1980. 2. Baskin, D. G.; Petracca, F.; Dorsa, D. M. Autoradiographic localization of specific binding sites for (3H) arginine vasopressin in the septum of the rat brain with tritium-sensitive film. Eur. J. Pharmacol. 90:155-157; 1983. 3. Bella, T.; Enyedi, P.; Spat, A.; Antoni, F. A. Pressor-type vasopressin receptors in the adrenal cortex: Properties of binding, effects on phosphoinositide metabolism and aldosterone secretion. Endocrinology 117:421-423; 1985. 4. Bepler, G.; Zeymer, U.; Mahmoud, S.; Fiskum, G.; Palaszynski, E.; Rotsch, M.; Willey, J.; Koros, A.; Cuttitta, F.; Moody, T. W. Substance P analogues function as bombesin receptor antagonists and inhibit small cell lung cancer clonal growth. Peptides 9:13671372; 1988. 5. Bunn, P. A., Jr.; Dienhart, D. G.; Chan, D.; Puck, T. T.; Tagawa, M.; Jewett, P. B.; Braunschweiger, E. Neuropeptide stimulation of calcium flux in human lung cancer cells: Delineation of alternative pathways. Proc. Natl. Acad. Sci. USA 87:2162-2166; 1990. 6. Choy, V. J.; Watldns, W. B. Maturation of the hypothalamoneurohypophysial system. 1. Localization of neurophysin, oxytocin and vasopressin in the hypothalamus and neural lobe of the developing rat brain. Cell Tissue Res. 197:325-336; 1979. 7. Dorsa, D. M.; Majumdar, L. A.; Petracca, F. M.; Baskin, D. G.; Cotnett, L. E. Characterization and localization of 3H-arginine vasopressin binding to rat kidney and brain tissue. Peptides 4:699-706; 1983. 8. Hadley, M. Endocrinology, 2nd ed. Englewood Cliffs, NJ: Prentice Hall, Inc.; 1988. 9. Jar& S.; Galliard, R. C.; Guillon, G.; Marie, J.; Schonenburg, P.; Muller, A. F.; Manning, M.; Sawyer, W. H. Vasopressin antagonists allow demonstration of a novel type of vasopressin receptor in the rat adenohypophysis. Mol. Pharmacol. 30:171-177; 1986. 10. Koob, G. F.; Dantzer, R.; Bluth, R. M.; Lebrun, C.; Bloom, F. E.; LeMoal, M. Central injections of arginine vasopressin prolong extinction of active avoidance. Peptides 7:213-218; 1986. 11. Kovacs, G. L.; Veldhuis, H. D.; Versteef, D. H. G.; DeWied, D. Facilitation of avoidance behavior by vasopressin fragments microinjected into limbic midbrain structures. Brain Res. 371:17; 1986. 12. Kruszynski, M.; Lammek, B.; Manning, M.; Seto, J.; Haldar, J.; Sawyer, W. H. (1-([3-Mercapto-13,[3-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine)arginine vasopressin and (l(13-mercapto13,[3-cyclopentamethylenepropionic acid)arginine vasopressin, two highly potent antagonists of the vasopressor response to arginine va-
sopressin. J. Med. Chem. 23:364-368; 1980. 13. Mahmoud, S. M.; Palaszynski, E.; Fiskum, G.; Coy, D. N.; Moody, T. W. Small cell lung cancer bombesin receptors are antagonized by reduced peptide bond analogues. Life Sci. 44:367-373; 1989. 14. Median, R.; Hsueh, A. J. W. Identification and characterization of arginine vasopressin receptor in the rat testis. Endocrinology 116: 416-423; 1985. 15. Moody, T. W.; Murphy, A.; Mahmoud, S.; Fiskum, G. Bombesinlike peptides elevate cytosolic calcium in small cell lung cancer cells. Biocbem. Biophys. Res. Commun. 147:189-195; 1987. 16. Oie, H. K., Brower, M.; Carney, D. N. Growth factor requirements for in vitro growth of endocrine and nonendocrine lung cancers in serum-free defined media. In: Becket, K.; Gazdar, A. F., eds. The endocrine lung in health and disease. Philadelphia: W. B. Sannders Co.; 1984:469--475. 17. Ruppert, S.; Scherer, G.; Schutz, G. Recent gene conversion involving bovine vasopressin and oxytocin precursor genes suggested by nucleotide sequence. Nature 308:554-557; 1984. 18. Sausville, E.; Carney, D. N.; Battey, J. The human vasopressin gene is linked to the oxytocin gene and is selectively expressed in a cultured lung cancer cell line. J. Biol. Chem. 260:10236-10241; 1985. 19. Shewey, L. M.; Dorsa, D. M. Vl-type vasopressin receptors in rat brain septum: Binding characteristics and the effects on inositol phospholipid metabolism. J. Neurosci. 8:1671-1677; 1988. 20. Staley, J.; Fiskum, G.; Davis, T. P.; Moody, T. W. Neurotensin elevates cytosolic calcium in small cell lung cancer cells. Peptides 10:1217-1221; 1989. 21. Staley, J.; Fiskum, G.; Moody, T. W. Cholecystokinin elevates cytosolic calcium in small cell lung cancer cells. Biochem. Biophys. Res. Commun. 163:605-610; 1989. 22. Staley, J.; Jensen, R. T.; Moody, T. W. CCK antagonists interact with CCK-B receptors on human small cell lung cancer cells. Peptides 11:1033-1036; 1990. 23. Swenson, R. R.; Beckwith, B. E.; Lamberty, K. J.; Krebs, S. J. Prenatal exposure to AVP or caffeine but not oxytocin alters learning in female rats. Peptides 11:927-932; 1990. 24. Van Wimersma Greidanus, T. J. B.; Van Ree, J. M.; de Wied, D. Vasopressin and memory. J. Pharmacol. Exp. Ther. 20:437-458; 1983. 25. Zachary, I.; Rozengurt, E. A substance P antagonist also inhibits specific binding and mitogenic effects of vasopressin and bombesinrelated peptides in Swiss 3T3 cells. Biochem. Biophys. Res. Commun. 137:135-141; 1986.
0196-9781/91 $3.00 + .00
Vasopressin Elevates Cytosolic Calcium in Small Cell Lung Cancer Cells I M. H O N G A N D T. W. M O O D Y 2
Department of Biochemistry and Molecular Biology, The George Washington University School of Medicine and Health Sciences, Washington, DC 20037 R e c e i v e d 28 M a r c h 1991 HONG, M. AND T. W. MOODY. Vasopressin elevates cytosolic calcium in small cell lung cancer cells. PEPTIDES 12(6) 1315-1319, 1991.--The ability of vasopressin to elevate cytosolic Ca2÷ in small cell lung cancer (SCLC) cells was investigated. Ten nanomolar vasopressin elevated the cytosolic Ca2÷ in 6 of 8 SCLC cell lines that were loaded with Fura-2 AM. Using SCLC cell line NCI-H345, the effect of vasopressin was dose dependent, being maximal at 100 nM, where the cytosolic Ca2÷ was elevated from 150 to 210 nM. Because addition of 1 mM EGTA had no effect on the vasopressin response, vasopressin released Ca2÷ from intracellular pools. Also, oxytocin weakly elevated the cytosolic Ca2+ . The response to vasopressin was strongly blocked by [(13-mercapto-13,13-cyclopentamethylene propionic acid)l,O-MeTyr2,ArgS]vasopressin and weakly blocked by [(13-mercapto-13,13-cyclopentamethylene propionic acid)l,O-MeTyr2,OmS]vasotocin. These data suggest that V l vasopressin receptors are present on SCLC cells. Small cell lung cancer
Vasopressin
Calcium
Fura-2
VASOPRESSIN (AVP) and oxytocin, which are nonapeptides, are synthesized in the paraventricular and supraoptic nuclei of the hypothalamus in the form of high molecular weight precursor proteins (6). After posttranslational processing, neurophysins and the 9 amino acid AVP and oxytocin result, which have 7 of the same 9 amino acid residues (17). These peptides function as posterior pituitary hormones and AVP regulates osmolarity, blood pressure and volume, whereas oxytocin induces uterine contractions and stimulates milk release from the mammary gland (8). Vasopressin is also active in the brain, where treatment of neonatal and adult animals affects learning and memory, respectively (23,24). After direct injection into the rat brain, AVP and [Cyt6]AVP(5-9) facilitate avoidance behavior (10,11). These actions may be mediated by AVP receptors, which have been detected in the CNS and periphery. The V 2 receptor, which is present in the kidney, stimulates adenylate cyclase activity (7). In contrast, the Via receptor, which is present in the liver, vascular smooth muscle, adrenals, testis and brain, increases phosphatidylinositol (PI) turnover (2, 3, 7, 14, 19). In the liver and hepatocyte, phospholipase C may be coupled to the V l receptor, resulting in inositol-l,4,5-trisphosphate (IP3) and increases in intracellular Ca 2+ . Hypophysial Via receptors also stimulate the PI pathway, resulting in elevated intracellular Ca 2 ÷ (9). The V~b receptor in the anterior pituitary is coupled to adenylate cyclase (9). Previously, immunoreactive AVP and AVP mRNA were reported to be present in some SCLC cell lines (18). In addition, AVP stimulates the clonal growth of certain SCLC cell lines (16). Here we investigated if AVP increased the cytoslic Ca 2÷
in Fura-2 AM-loaded SCLC cells. Structure-activity studies using AVP analogues suggest that V~ receptors are present in SCLC cell line NCI-H345. METHOD The human tumor cell lines were cultured in serum-supplemented medium (RPMI 1640 containing 10% heat-inactivated fetal calf serum) in a humidified atmosphere of 5% CO 2 and 95% air at 37°C. On the day of the assay, cells were harvested by centrifugation at 1000 x g for 10 minutes. Cells were washed and resuspended in assay buffer (SIT medium-RPMI 1640 conmining 3 x 10 - 8 M Na2SeO 3, 5 ixg/ml bovine insulin, and 10 p,g/ml human transferrin). Here we investigated if AVP analogues altered the intracellular Ca 2÷ . For the cytosolic Ca 2 + assays, cell line NCI-H345 was harvested 24 hours after a medium change, washed, and resuspended in SIT media containing 20 mM HEPES/NaOH (pH 7.4). SCLC cells ( 2 . 5 x 106/ml) were incubated with 5 IxM Fura-2 AM (Calbiochem Inc., La Jolla, CA) at 370(2 for 30 minutes in a shaking water bath. Cells were centrifuged at 1 5 0 × g for 2 minutes, and the pellet containing SCLC cells loaded with Fura-2 were resuspended to the same concentration. Fluorescence was monitored via a spectrofluorometer at an excitation and emission wavelength of 340 nm and 510 nm, respectively. The spectrofluorometer was equipped with a temperatureregulated cuvette holder in addition to a magnetic stirrer. Cytosolic Ca 2 + was measured upon addition of various AVP analogs and antagonists.
IPortions of this article were presented at the 12th Annual Winter Neuropeptide Conference, Breckenridge, CO (February, 1991). 2Requests for reprints should be addressed to Dr. Terry W. Moody, GWU Biochemistry Department, 2300 Eye St. N.W., Washington, DC 20037.
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TABLE 1 EFFECT OF PEPTIDES OF C a 2+ LEVELS IN SCLC CELLS Cell Line
AVP
BN
CCK
NT
NCI-H146 NCI-H187 NCI-H209 NCI-H345 NCI-N417 NCI-H510 NCI-H1092 NCI-H 1694
-+ ++ ++ ++ ++ + .
-+ ++ ++ -++ +
++ + ++ ++ -++ +
-++ ++ ++ --+
.
.
.
Peptides were added at a 10 nM concentration and the Ca2+ response determined in the indicated SCLC cell line. Strong response, + +; weak response, +; no response, --.
RESULTS Previously, we found that neurotensin (NT), bombesin/gastrin releasing peptide (BN/GRP) and cholecystokinin (CCK) elevated cytosolic Ca 2÷ in SCLC cell line NCI-H345 via distinct neuropeptide receptors (15, 20, 21). Table 1 shows that AVP caused a strong Ca 2 ÷ response in 4 of 8 SCLC cell lines examined, including NCI-H209, H345, N417 and H510. In contrast, 10 nM AVP caused a weak response in 2 other cell lines (NCIH187 and H1092), whereas there was no response in 2 other cell lines (NCI-H146 and H1694). Also, CCK, BN and NT caused a Ca 2 ÷ response in 6, 5 and 4 cell lines, respectively, of the 8 cell lines examined. Subsequently, the effect of AVP was investigated using cell line NCI-H345, which had a strong Ca 2 ÷ response to AVP, BN, CCK and NT. Figure 1 shows that AVP elevated the cytosolic Ca 2÷ in a dose-dependent manner. The cytosolic Ca 2 ÷ was minimally affected by 0.1 or 1 nM AVP, whereas 10 nM AVP increased the cytosolic Ca 2÷ from 150 to 190 nM. At 10 nM AVP, the cytosolic Ca 2÷ increased slowly, reaching maximal values after 30 s. Then after a transient steady state, the cytosolic Ca 2 ÷ slowly returned to basal levels within 5 min. At a 100 or 1000 nM
A
0.1 nM AVP
B
1 nM AVP
AVP concentration, the cytosolic Ca 2+ increased from 150 to 210 nM. AVP released Ca 2+ from intracellular organelles. Figure 2 shows that when 10 nM AVP was added to cell line NCI-H345, which was loaded with Fura-2 AM, there was an increase in the fluorescence intensity. When 1 mM EGTA was added to the extracellular medium, there was a sharp reduction in the fluorescence intensity due to extracellular Fura-2 AM going from the Ca 2+ bound to Ca 2÷ free state. When 10 nM AVP was subsequently added, there was still an increase in the fluorescence intensity almost identical to that observed previously. These data suggest that AVP does not open extracellular Ca 2+ channels. Oxytocin, which is structurally similar to AVP, had no effect on the cytosolic Ca 2+ at a 10 nM dose (data not shown). At 100 nM oxytocin, the cytosolic Ca 2+ increased from 150 to 180 nM. At a 1000 nM or 10,000 nM concentration, oxytocin maximally increased the cytosolic Ca 2+ . These data indicate that oxytocin is over 1 order of magnitude weaker at elevating the cytosolic Ca 2 + than is AVP. Oxytocin may elevate cytosolic Ca 2÷ by interacting with AVP receptors. Figure 3B shows that when 10 p,M oxytocin was added, a strong Ca 2+ response was obtained. If 10 nM AVP was subsequently added, the Ca 2+ response was absent. These data indicate that oxytocin and AVP elevate cytosolic Ca 2+ through a similar mechanism. Similarly, if 10 nM AVP was added there was a strong Ca 2+ response (Fig. 3A). If 10 nM AVP was subsequently added, the response was absent, possibly due to receptor desensitization. In contrast, BN, CCK or NT elevated the cytosolic Ca 2 ÷ but had no effect on the AVP Ca 2 + response, whereas pressinoic acid was inactive (data not shown). [D-Argl,D-Pro2,D-Trp7'9,Leull]substance P ([APTTL]SP) was an antagonist in that, if 10 IxM [APTTL]SP was added, there was no Ca 2÷ response (Fig. 3C). If 10 nM AVP was subsequently added, there was no increase in the cytosolic Ca 2 ÷. In addition to [APTTL]SP, [(13-mercapto-13,13-cyclopentam e t h y l e n e propionic a c i d ) l , O - M e T y r 2 , o r n i t h i n e a ] v a s o t o c i n [d(CH2)5,Tyr(Me)2]OVT functioned as a weak antagonist. [d(CH2)5,Tyr(Me)2]OVT (1000 nM) had no effect on the cytosolic Ca 2÷' but decreased the increase in cytosolic Ca 2+ caused by 1 nM AVP (Fig. 4). If 10 nM AVP was subsequently added, there was a slight increase in the cytosolic Ca 2÷ from 150 to 160 nM. If 1000 nM AVP was subsequently added,
C
D
10 nM AVP
100 nM AVP
E
1000 nM AVP
.Z.__
20C
15o
~--- 4 rain-~
~--- 4 rain--~
~--- 4 min-~
~- 4 rain--~
4 rain - - ~
Time, min
FIG. 1. Effect of vasopressin (AVP) on cytosolic Ca2+ . SCLC cell line NCI-H345 was loaded with Fura-2 AM and the ability of (A) 0.1 nM AVP, (B) 1 nM AVP, (C) 10 nM AVP, (D) 100 nM AVP and (E) 1000 nM AVP to elevate cytosolic Ca2+ determined. This experiment is representative of 3 others.
VASOPRESSIN AND SCLC
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10 nM VP
E
~
o
=;
4 min
c o
10 nM VP
u.
~9---- 4 rain ----~ Time, min
FIG. 2. Effect of EGTA on AVP response. The ability of 10 nM AVP to elevate the cytosolic Ca 2÷ was determined prior to and after the addition of 1 mM EGTA to the extracellular medium.
there was a strong C a 2+ response. These data indicate that [d(CH2)5,Tyr(Me)2]OVT is a reversible AVP antagonist. [(13-Mercapto-13,13-cyclopenthamethylene propionic acid)l,O MeTyr2]AVP [d(CH2)5,T~yr(Me)2]AVP was a strong AVP antagonist. [d(CH2)5,Tyr(Me)~]AVP (1 nM) had no effect on the cytosolic Ca 2+ , but decreased the amplitude of the Ca 2+ response to 10 nM AVP (Fig. 5A, B). [d(CH2)5,Tyr(Me)2]AVP (100 nM) had no effect on the cytosolic Ca 2÷ , but totally antagonized the response to 10 nM AVP (Fig. 5C). Similarly, 10 nM [d(CH2)5,Tyr(Me)2]AVP blocked the response to 10 nM AVP, whereas 10 nM [d(CH2)5,Tyr(Me)2]OVT had no effect on the response to 10 nM AVP (data not shown). These data indi-
cate that [d(CH2)5,Tyr(Me)2]AVP is approximately 2 orders of magnitude more potent than is [d(CH2)5,Tyr(Me)2]OVT as a AVP antagonist. DISCUSSION
Previously, 100 nM AVP was reported to elevate the cytosolic Ca 2+ in SCLC cell line NCI-H345 and H510, but not large cell cancer cell line NCI-H460 or breast cancer cell lines ZR-75 or MCF-7 (5). Further, the Ca 2+ response to AVP was distinct from that of BN, NT, CCK or bradykinin. Here the pharmacology of the AVP Ca 2 ÷ response was investigated.
C
10 nM AVP
200
150
10 nM AVP
,l
l
f-...__ L I-
10 uM Oxy 10 nMAVP 10 uM (APTTL)SP 10 nM AVP
4 rain - ~
!
~1---- 4 min - - ~
~1--- 4 min
~t -[
Time, min FIG. 3. Pharmacologic specificity of the AVP response. The cytosolic Ca 2 + levels were determined after the addition of (A) 10 nM AVP followed by 10 nM AVP, 03) 10 p,M oxytocin (Oxy) followed by 10 nM AVP and (C) 10 p,M [ A F I ' ~ ] S P followed by 10 nM AVP.
1318
HONG AND MOODY
1 uM
1 nM
(d(CH2)5,
AVP
]Tyr(Me)2)OVT
10 nM AVP
1000 nM AVP
i
,l 200 4-
150
I_
lO mi.
I-
_1
-I
Time, rain
FIG. 4. Reversibility of an AVP antagonist. [d(CH2)5,Tyr(Me)2]OVT (1 ~M) had no effect on the cytosolic Ca2÷ . AVP was subsequently added at a 1 nM concentration, followed by 10 nM AVP and 1000 nM AVP.
Ca 2+ response caused by 10 nM AVP. These data suggest that the AVP, GRP and CCK-B receptors present on SCLC cell line NCI-H345 are distinct. AVP may elevate the cytosolic Ca 2+ on SCLC cell line NCI-H345 by binding to Via receptors. The EDso for AVP and oxytocin to elevate the cytosolic Ca 2÷ is approximately 5 and 200 nM, respectively. Further, the EDso for [d(CH2)5,Tyr (Me)2]AVP and [d(CH2)5,Tyr(Me)2]OVT to inhibit the increase in cytosolic Ca 2+ caused by 10 nM AVP is approximately 1 and 1000 nM, respectively. Previously, [d(CH2)5,Tyr(Me)2]AVP and [d(CH2)5,Tyr(Me)2]OVT were found to be V] and V 2 receptor antagonists (1,9). For V 1 receptors in the rat septum, the K i values for vasopressin, oxytocin and [d(CH2)5,Tyr(Me)z]AVP to inhibit 3H-AVP binding are 2, 185 and 1 nM, respectively. Previously, it was found that [APTTL]SP, a SP antagonist, also antagonizes SCLC BN/GRP receptors. Thus 10 p,M [APTTL]SP inhibited ~2~I-GRP binding to NCI-H345, the cytosolic Ca 2 ÷ induced by 10 nM BN and clonal growth (4). Also, 10 txM [APTTL]SP inhibited 125I-GRP and 3H-AVP binding to
Ten nM AVP increased the cytosolic Ca 2 + in 6 of 8 SCLC cell lines examined. These data suggest that many, but not all, SCLC cells have functional AVP receptors. Cell lines NCI-H209 and NCI-H345 strongly responded to AVP as well as BN, CCK and NT. Using cell line NCI-H345, however, the mechanisms by which AVP, BN, CCK and NT elevated cytosolic Ca 2÷ were different. If 10 nM AVP was added, there initially was an increase in the cytosolic Ca 2+ from 150 to 190 nM within 30 s, followed by a slow decrease to baseline over the next 5 min. If 10 nM AVP or 10 IxM oxytocin was subsequently added, there was a lack of Ca 2 ÷ response. These data suggest that AVP or oxytocin increase the cytosolic Ca 2 ÷ through a similar mechanism. AVP and oxytocin do not deplete the cell of Ca 2+ because addition of 10 nM, BN, NT or CCK after the addition of 10 nM AVP caused a strong increase in the cytosolic Ca 2÷ . Therefore, AVP and oxytocin may bind to the same receptor. Further, the cytosolic Ca 2÷ response to BN is blocked by [Psila'14,Leul4]BN (13) and the response to CCK blocked by L-365,718 (22), and neither of these compounds inhibits the
A
B
C
I nM (d(CH2)5, 10 nM AVP 20O
Tyr(Me}'2)AVP
100 nM (d(CH2]5, 10 nM AVP
Tyr(Me)2)AVP 10 nM AVP
1
r2
Time, mtn
FIG. 5. Dose response of a AVP antagonist. The cytosolic C a 2 + response was determined after the addition of (A) 10 nM AVP, (B) 1 nM [d(CH2)~,Tyr(Me)2]AVP followed by 10 nM AVP and (C) 100 nM [d(CH2)5,Tyr(Me)2]AVP followed by 10 nM AVP.
VASOPRESSIN AND SCLC
1319
Swiss 3T3 cells, the increase in cytosolic Ca 2 + caused by AVP and BN as well as the increase in 3H-thymidine uptake induced by AVP or BN (25). Because the SP antagonist bears little sequence homology to AVP or BN, it may interact with guanine nucleotide binding proteins impairing the receptor conformation. It remains to be determined if AVP is an autocrine growth factor for SCLC cells. Because the peptide is synthesized and translated in SCLC cells, and is biologically active, it could be an autocrine growth factor. Besides elevating cytosolic Ca 2+ , AVP (10 ng/ml) stimulated the growth of SCLC cells
(16). It remains to be determined if AVP antagonists such as [d(CH2)5,Tyr(Me)2]AVP inhibit the growth of SCLC. In summary, AVP elevates cytosolic Ca 2+ in some SCLC cell lines. Structure-activity studies suggest that V 1 receptors may be present on SCLC. AC~O~E~E~S The authors thank J. Staley for technical assistance and Dr. G. Fiskum for helpful discussions. Supported in part by NCI grant CA-53477.
REFERENCES 1. Bankowitz, K.; Manning, M.; Seto, J.; Haldar, J.; Sawyer, W. H. Design and synthesis of potent in vivo antagonists of oxytocin. Int. J. Pept. Protein Res. 16:382-391; 1980. 2. Baskin, D. G.; Petracca, F.; Dorsa, D. M. Autoradiographic localization of specific binding sites for (3H) arginine vasopressin in the septum of the rat brain with tritium-sensitive film. Eur. J. Pharmacol. 90:155-157; 1983. 3. Bella, T.; Enyedi, P.; Spat, A.; Antoni, F. A. Pressor-type vasopressin receptors in the adrenal cortex: Properties of binding, effects on phosphoinositide metabolism and aldosterone secretion. Endocrinology 117:421-423; 1985. 4. Bepler, G.; Zeymer, U.; Mahmoud, S.; Fiskum, G.; Palaszynski, E.; Rotsch, M.; Willey, J.; Koros, A.; Cuttitta, F.; Moody, T. W. Substance P analogues function as bombesin receptor antagonists and inhibit small cell lung cancer clonal growth. Peptides 9:13671372; 1988. 5. Bunn, P. A., Jr.; Dienhart, D. G.; Chan, D.; Puck, T. T.; Tagawa, M.; Jewett, P. B.; Braunschweiger, E. Neuropeptide stimulation of calcium flux in human lung cancer cells: Delineation of alternative pathways. Proc. Natl. Acad. Sci. USA 87:2162-2166; 1990. 6. Choy, V. J.; Watldns, W. B. Maturation of the hypothalamoneurohypophysial system. 1. Localization of neurophysin, oxytocin and vasopressin in the hypothalamus and neural lobe of the developing rat brain. Cell Tissue Res. 197:325-336; 1979. 7. Dorsa, D. M.; Majumdar, L. A.; Petracca, F. M.; Baskin, D. G.; Cotnett, L. E. Characterization and localization of 3H-arginine vasopressin binding to rat kidney and brain tissue. Peptides 4:699-706; 1983. 8. Hadley, M. Endocrinology, 2nd ed. Englewood Cliffs, NJ: Prentice Hall, Inc.; 1988. 9. Jar& S.; Galliard, R. C.; Guillon, G.; Marie, J.; Schonenburg, P.; Muller, A. F.; Manning, M.; Sawyer, W. H. Vasopressin antagonists allow demonstration of a novel type of vasopressin receptor in the rat adenohypophysis. Mol. Pharmacol. 30:171-177; 1986. 10. Koob, G. F.; Dantzer, R.; Bluth, R. M.; Lebrun, C.; Bloom, F. E.; LeMoal, M. Central injections of arginine vasopressin prolong extinction of active avoidance. Peptides 7:213-218; 1986. 11. Kovacs, G. L.; Veldhuis, H. D.; Versteef, D. H. G.; DeWied, D. Facilitation of avoidance behavior by vasopressin fragments microinjected into limbic midbrain structures. Brain Res. 371:17; 1986. 12. Kruszynski, M.; Lammek, B.; Manning, M.; Seto, J.; Haldar, J.; Sawyer, W. H. (1-([3-Mercapto-13,[3-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine)arginine vasopressin and (l(13-mercapto13,[3-cyclopentamethylenepropionic acid)arginine vasopressin, two highly potent antagonists of the vasopressor response to arginine va-
sopressin. J. Med. Chem. 23:364-368; 1980. 13. Mahmoud, S. M.; Palaszynski, E.; Fiskum, G.; Coy, D. N.; Moody, T. W. Small cell lung cancer bombesin receptors are antagonized by reduced peptide bond analogues. Life Sci. 44:367-373; 1989. 14. Median, R.; Hsueh, A. J. W. Identification and characterization of arginine vasopressin receptor in the rat testis. Endocrinology 116: 416-423; 1985. 15. Moody, T. W.; Murphy, A.; Mahmoud, S.; Fiskum, G. Bombesinlike peptides elevate cytosolic calcium in small cell lung cancer cells. Biocbem. Biophys. Res. Commun. 147:189-195; 1987. 16. Oie, H. K., Brower, M.; Carney, D. N. Growth factor requirements for in vitro growth of endocrine and nonendocrine lung cancers in serum-free defined media. In: Becket, K.; Gazdar, A. F., eds. The endocrine lung in health and disease. Philadelphia: W. B. Sannders Co.; 1984:469--475. 17. Ruppert, S.; Scherer, G.; Schutz, G. Recent gene conversion involving bovine vasopressin and oxytocin precursor genes suggested by nucleotide sequence. Nature 308:554-557; 1984. 18. Sausville, E.; Carney, D. N.; Battey, J. The human vasopressin gene is linked to the oxytocin gene and is selectively expressed in a cultured lung cancer cell line. J. Biol. Chem. 260:10236-10241; 1985. 19. Shewey, L. M.; Dorsa, D. M. Vl-type vasopressin receptors in rat brain septum: Binding characteristics and the effects on inositol phospholipid metabolism. J. Neurosci. 8:1671-1677; 1988. 20. Staley, J.; Fiskum, G.; Davis, T. P.; Moody, T. W. Neurotensin elevates cytosolic calcium in small cell lung cancer cells. Peptides 10:1217-1221; 1989. 21. Staley, J.; Fiskum, G.; Moody, T. W. Cholecystokinin elevates cytosolic calcium in small cell lung cancer cells. Biochem. Biophys. Res. Commun. 163:605-610; 1989. 22. Staley, J.; Jensen, R. T.; Moody, T. W. CCK antagonists interact with CCK-B receptors on human small cell lung cancer cells. Peptides 11:1033-1036; 1990. 23. Swenson, R. R.; Beckwith, B. E.; Lamberty, K. J.; Krebs, S. J. Prenatal exposure to AVP or caffeine but not oxytocin alters learning in female rats. Peptides 11:927-932; 1990. 24. Van Wimersma Greidanus, T. J. B.; Van Ree, J. M.; de Wied, D. Vasopressin and memory. J. Pharmacol. Exp. Ther. 20:437-458; 1983. 25. Zachary, I.; Rozengurt, E. A substance P antagonist also inhibits specific binding and mitogenic effects of vasopressin and bombesinrelated peptides in Swiss 3T3 cells. Biochem. Biophys. Res. Commun. 137:135-141; 1986.
