Spiperone: Evidence for Uptake Into Secretory Granules (Pituitary/Prolactin/Biogenic Amine/Reserpine) PRISCILLA S

Proc. Nati. Acad. Sci. USA Vol. 81, pp. 1867-1870, March 1984 Neurobiology Spiperone: Evidence for uptake into secretory granules (pituitary/prolactin/biogenic amine/reserpine) PRISCILLA S. DANNIES, MARLA S. RUDNICK, HANA FISHKES, AND GARY RUDNICK Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510 Communicated by Aaron B. Lerner, November 21, 1983

ABSTRACT Spiperone, a dopamine antagonist widely mmol, or Amersham, with a specific activity of 16 Ci/mmol used as a specific ligand for dopamine and serotonin receptors, (1 Ci = 37 GBq). Monensin and reserpine were from Calbio- is actively accumulated into the F4C1 strain of rat pituitary chem. Benztropine was from Merck, Sharp and Dohme. tumor cells. The accumulation of 10 nM [3Hjspiperone was Cell Culture. The F4C1 cell strain is a clonal cell line from a linear for 3 min and reached a steady state after 10 min. Spi- rat pituitary tumor that makes prolactin and growth hormone perone accumulation was reduced 50% by preincubation with (9). The cells were obtained from Ingrid Richardson (Har- 5 jiM reserpine, an inhibitor of biogenic amine transport into vard School of Public Health). They were cultured on 35-mm secretory granules, and was also blocked by monensin and am- plates in 1.0 ml of 1:1 mixture of Dulbecco's minimal essen- monium chloride, both of which increase the pH of intracellu- tial medium and Ham's nutrient mixture F10 plus 15% horse lar storage organelles. Uptake was not affected by replacing serum. Cultures were maintained in a humidified atmo- sodium in the buffer with lithium at equimolar concentrations. sphere of 95% air/5% CO2 at 37°C. Cells were cultured at a Spiperone at 1 jiM inhibited by over 50% serotonin transport density of 5 x 104 cells per plate. The medium was changed 2 into membrane vesicles isolated from platelet dense granules; days later, and uptake experiments were performed the next this concentration inhibited the Na'-dependent plasma mem- day. brane transport system less than 10%. The data indicate spi- [3H]Spiperone Uptake. Unless otherwise indicated, the perone specifically interacts with the secretory granule amine plates were rinsed twice at the start of the experiment with 1 transport system and suggest that this transport system is ml of buffer per rinse. Buffer used for the uptake experi- found in the F4C1 pituitary cell strain as well as in platelets and ments contained 120 mM NaCl, 5 mM Hepes, 5 mM KCl, 2 neurons. The data also suggest that experiments utilizing spi- mM MgCl2, and 10 mM glucose, pH 7.2. After rinsing, the perone to measure dopanine and serotonin receptors be inter- cells were incubated at 37°C with 1 ml of buffer containing preted with caution. [3H]spiperone and other drugs as indicated. The uptake period was ended by rinsing the cells three times with cold saline Prolactin production by the anterior pituitary gland in the rat and then dissolving the cells in 1.0 ml of 0.1 M NaOH. The is primarily under negative control by the hypothalamus (1). radioactivity of a 0.4-ml aliquot was measured in Biofluor At least part of the inhibition of prolactin secretion appears scintillation fluid (New England Nuclear), and 0.4 ml was to be caused by dopamine released from the hypothalamus. used to determine cell number or protein content. Protein Dopamine acts directly on the anterior pituitary gland to in- determinations were more easily reproducible than cell hibit prolactin production (2), and measurements of dopa- counts and were routinely used; we found that 1 mg of pro- mine in the portal blood system connecting the hypothala- tein is equivalent to 9 x 105 cells and used this factor to mus to the anterior pituitary gland have shown that the con- convert protein to cell number. Protein was determined by centration is sufficient to cause significant inhibition of the Lowry technique (10), using bovine serum albumin as a prolactin release (3). standard. The pituitary gland contains dopamine-binding sites, and Prolactin Determinations. Prolactin was assayed by micro- the relative affinities of agents for these sites correlate with complement fixation as described (11). their relative abilities to inhibit prolactin release (4, 5). Serotonin Transport Assays. Serotonin transport into [3H]Spiperone, a dopaminergic antagonist, has been used to membrane vesicles isolated from platelet dense granules was estimate the distribution of these binding sites (6). This com- measured at 37°C as described (12). Transport into platelet pound is also used frequently to measure both dopaminergic plasma membrane vesicles was measured at 25°C as de- and serotonergic receptors in the brain (7, 8). The pituitary scribed (13). gland is a heterogeneous population of cell types; studies of RESULTS biochemical mechanisms of dopaminergic action are, therefore, difficult to interpret. We have been screening pituitary F4C1 Cells Take Up Spiperone. Data in Fig. 1 show the cell lines for the presence of dopamine receptors, to have a uptake of 10 nM [3H]spiperone into F4C1 cells. The accumu- pure population of prolactin-producing cells. During the lation was linear in this and other experiments for at least 3 course of these investigations we found evidence that sug- min; after this time the rate of accumulation decreased. In gests a prolactin-producing tumor cell strain, F4, may accu- other experiments we have found accumulation at the end of mulate [3H]spiperone by a transport system similar to that 1 hr was no greater than after 10 min of incubation. Charac- found in secretory granules and synaptic vesicles and dis- teristics of this uptake were different than those of spiperone tinct from binding to dopamine receptors. binding to the dopaminergic plasma membrane receptors. First, binding to these receptors is half-maximally saturated METHODS at concentrations less than 1 nM (6), but uptake of [3H]spiperone into F4C1 cells was proportional to the amount of Materials. [benzene ring-3H]Spiperone was obtained from [3H]spiperone in the medium to concentrations of at least 30 New England Nuclear, with a specific activity of 19.9 Ci/ nM (Fig. 2). Second, 10 ,uM dopamine and 3 nM bromocriptine, a dopaminergic agonist, inhibited binding of 1 nM The publication costs of this article were defrayed in part by page charge [3H]spiperone to the dopaminergic receptor by more than payment. This article must therefore be hereby marked "advertisement" 50% (6). We found the uptake of 0.3 nM [3H]spiperone in in accordance with 18 U.S.C. §1734 solely to indicate this fact. F4C1 cells was not affected by concentrations of dopamine

1867 Downloaded by guest on September 29, 2021 1868 Neurobiology: Dannies et al. Proc. Natl. Acad Sci. USA 81 (1984)

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FIG. 1. Accumulation of 10 nM [3H]spiperone into cells with FIG. 3. Effect of sodium on [3H]spiperone uptake. F4C1 cells time. were incubated with 10 nM [3H]spiperone in buffer containing 120 mM NaCl (o) or 120 mM LiCI (e) for the indicated times. ranging from 1 to 400 ,uM or by bromocriptine in concentrations ranging from 4 nM to 1 AM (data not shown). Therefore F4C1 cells (Fig. 5); 10 mM NH4Cl and 10 ,uM monensin each the uptake of spiperone is not caused by binding to the previ- cause over 50% inhibition of uptake. Reserpine, monensin, ously characterized membrane receptor. and ammonium chloride do not appear to act by toxic effects Characterization of Uptake. Two types of amine transport on the cells for two reasons: first, there is no effect on cell systems have been characterized in neurons and other secre- number or the amount of cell protein during the period the tory cells. Na+-dependent plasma membrane transporters cells are incubated with these agents, and second, there is are specific for the transported substrate and are inhibited by little effect on cell protein and prolactin accumulation in the compounds such as benztropine (14) and imipramine (15). medium 4 days after treatment with these agents (Table 2). Granule transporters are driven by the transmembrane H+ To further test the possibility that spiperone interacts with gradient, are less specific, and are inhibited by reserpine the transport system in secretory granules, we measured the (16). Replacing sodium in the buffer with lithium did not af- ability of spiperone to inhibit serotonin transport into mem- fect [3H]spiperone uptake by F4C1 cells (Fig. 3). Uptake was brane vesicles isolated from platelet dense granules. These markedly affected only at lM10 benztropine (Table 1), a vesicles accumulate serotonin to high internal concentra- concentration that is high compared to concentrations of less tions when supplied with ATP as an energy source. This ac- than 1 AM that inhibit the plasma membrane dopamine trans- cumulation is sensitive to inhibition by reserpine and uncou- port system (14). plers, indicating that it is a good model of biogenic amine Reserpine inhibits amine uptake by the transport system in accumulation in many secretory granules (12, 18). As shown secretory vesicles (16). Reserpine at 5 ,u M also inhibits accu- in Fig. 6, spiperone is a potent inhibitor of this serotonin mulation of [3H]spiperone into F4C1 cells by 50% (Fig. 4). transport system. Transport is more than 50% inhibited at 1 -Amine transport into secretory vesicles is driven by a pH ,M spiperone and almost completely blocked at 5 ,uM. gradient. Thus two agents that destroy this gradient, monen- As a control, we tested spiperone's ability to inhibit sero- sin and ammonium chloride, inhibit amine transport (17). tonin transport into platelet plasma membrane vesicles. This These agents also inhibit [3H]spiperone accumulation into transport system is insensitive to reserpine or ATP but is driven by transmembrane gradients of Na+, K+, and Cl- and is blocked by imipramine (18). As shown in Fig. 6, this transport system is much less sensitive to inhibition by spiperone. At 1 ,uM spiperone, the plasma membrane transport 20 / system retains almost 90% of its activity, whereas the granule transport system was over 75% inhibited. Thus, spiperone specifically inhibits biogenic amine transport into secretory granules, as would be expected for a substrate of the z:: 15 transport system. 0 DISCUSSION I.. We propose, on the basis of the data presented in this paper, C0CP~~~ that in addition to binding at dopamine and serotonin receptors, spiperone is actively accumulated by some cells. In 00

5 / Table 1. Effect of benztropine on [3H]spiperone uptake Benztropine, [3H]Spiperone uptake, AuM pmol/mg protein 0 2.2 ± 0.1 10 20 30 1 1.9 ± 0.1 [3H] Spiperone, nM 10 1.1 ± 0.1

FIG. 2. Effect of spiperone concentration on uptake. F4C1 cells Cells were incubated with benztropine at the indicated concentra- the mean ± were incubated for 2 min with the concentrations of [3H]spiperone tions and 10 nM [3H]spiperone for 3 min. Each value is indicated on the abscissa. the range of values from duplicate plates. Downloaded by guest on September 29, 2021 Neurobiology: Dannies et aL Proc. Natl. Acad. Sci. USA 81 (1984) 1869

Table 2. Effect of incubation with reserpine, monensin, or ammonium chloride on prolactin production and cell protein Protein, Prolactin, Treatment mg per plate pg/mg protein per 4 days 0500 Reserpine, 5 AtM 5.8 ± 0.61 0.051 ± 0.005 Control 7.6 0.78 0.036 0.006 0.2 ± ± 0 C~~~~~~~ Monensin, 5 AuM 7.3 ± 1.45 0.041 ± 0.008 :3 O Control 7.1 ± 1.10 0.042 ± 0.008 'C0.6 E~. ;, 0 0 NH4Cl, 5 mM 6.9 ± 1.29 0.043 ± 0.006 Control 7.2 ± 1.16 0.040 ± 0.007 0.2- Cells were rinsed and treated with the compounds in buffer, using the same procedure as we used to inhibit spiperone uptake (30 min of incubation with reserpine and 2 min with monensin and ammoni- 0 2.5 5 10 20 um chloride). Then the buffer was replaced with medium and the Reserpine, pM cells were grown for 4 days. Medium was collected and prolactin in the medium was measured. Each value is the mean ± SD of tripli- FIG. 4. Effect of reserpine on [3H]spiperone uptake in F4C1 cate plates. cells. At the start of the experiment, cells were rinsed once and then buffer with the indicated concentration of reserpine was added. The neurons is that prolactin cells have spontaneous action po- cells were incubated for 30 min at 370C, the buffer was replaced with tentials (20); agents that trigger release of prolactin can stim- fresh buffer containing reserpine at the indicated concentrations plus 10 nM [3H]spiperone, and the cells were incubated for 3 min ulate an increase in the frequency of these action potentials, before they were collected. Reserpine inhibition was less effective while agents that inhibit prolactin release inhibit generation without the preincubation period. of these potentials. The reserpine-sensitive spiperone uptake in the F4C1 cells indicates that these pituitary tumor cells may share another trait with neurons, the biogenic amine F4C1 cells spiperone apparently diffuses through the plasma transport system of the secretory vesicles. membrane and then is transported into the secretory gran- Normal prolactin-producing cells may have both the plas- ules by a biogenic amine transport system. Diffusion back ma membrane and the granule membrane transport system. through the secretory granule membranes of F4C1 cells must The reason for suggesting normal cells may have both sys- not occur as rapidly as transport into the vesicles, since we tems is that Porter and co-workers have demonstrated detect accumulation. F4C1 cells evidently lack functional [3H]dopamine accumulation in intact cells; the dopamine plasma membrane amine transport systems for dopamine or was found in a fraction with the same density as a fraction serotonin, since we did not detect sodium-dependent dopa- that contained the majority of the intracellular prolactin (21). mine or serotonin uptake into these cells (unpublished re- They therefore suggested dopamine was accumulating in the sults). Therefore we could not test in these intact cells secretory granules. Because they found that 15 uM cis-flu- whether spiperone could inhibit amine transport. We did, penthixol (a dopaminergic antagonist) and 10 ,uM perebedil however, demonstrate that spiperone directly inhibits sero- (an agonist) prevented the accumulation of dopamine, they tonin uptake into vesicles isolated from platelet granules. Al- suggested that the accumulation was a receptor-mediated though platelet granule vesicles fail to accumulate spiperone event (22). If, however, dopaminergic agents also compete (data not shown) this may be due to faster efflux from these with dopamine for uptake into granules, then dopamine ac- vesicles than from F4C1 granules. cumulation would be decreased. We suggest that dopamine Fujita et al. (19) have drawn attention to the similarity of accumulation in the pituitary gland may be inhibited by function and morphology of neuronal cells and endocrine agents that inhibit the granule transport system. cells such as prolactin-secreting cells. They have called these endocrine cells paraneurons. A further resemblance to

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0 i... 0O 0 0.1 1.0 10 0 0.1 10 1 2 3 4 5 6 7 8 9 10 NH4CI, mM Monensin, ,pM Spiperone, pM FIG. 5. Effects of ammonium chloride (A) and monensin (B) on FIG. 6. Spiperone inhibits secretory granule biogenic amine uptake of [3H]spiperone uptake by F4C1 cells. Cells were treated transport. Accumulation of [3H]serotonin by membrane vesicles de- with 20 nM [3H]spiperone plus monensin or ammonium chloride at rived from platelet dense granules (o) and platelet plasma membrane the indicated concentration for 2 min. No preincubation was neces- (o) was measured in the presence of spiperone at the indicated con- sary to see inhibition. centrations. Downloaded by guest on September 29, 2021 1870 Neurobiology: Dannies et al. Proc. NatL Acad Sci. USA 81 (1984) In many studies, association of a radioactive ligand with 7. List, S. J. & Seeman, P. (1981) Proc. Nati. Acad. Sci. USA 78, cells or membranes is interpreted as binding to surface re- 2620-2624. ceptors and not accumulation within the cell or vesicle. The 8. Withy, R. M., Mayer, R. J. & Strange, P. G. (1981) J. Neuro- results presented here indicate that at least one of these li- chem. 37, 1144-1154. 9. Richardson, U. I. (1976) J. Cell Physiol. 88, 287-296. gands accumulates intracellularly via cellular transport sys- 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, tems. We therefore urge caution in interpreting ligand asso- R. J. (1951) J. Biol. Chem. 193, 265-275. ciation as binding without considering transport as an alter- 11. Tashjian, A. H., Jr., Bancroft, F. C. & Levine, L. (1970) J. native mechanism. Cell. Biol. 47, 61-70. 12. Fishkes, H. & Rudnick, G. (1982) J. Biol. Chem. 257, 5671- We thank Dr. Ingrid Richardson of Harvard School of Public 5677. Health for the F4C1 cells. This work was supported by United States 13. Rudnick, G. (1977) J. Biol. Chem. 252, 2170-2174. Public Health Service Grants HD-11487 and HL-21217. P.S.D. is a 14. Koe, K. B. (1976) J. Pharmacol. Exp. Ther. 199, 649-661. recipient of Research Career Development Award HD-00272, and 15. Shaskan, E. G. & Snyder, S. H. (1970) J. Pharmacol. Exp. G.R. is an Established Investigator of the American Heart Associa- Ther. 175, 404-418. tion. 16. Stitzel, R. (1977) Pharmacol. Rev. 28, 179-205. 17. Njus, D., Knoth, J. & Zallakian, M. (1981) in Current Topics in Bioenergetics, ed. Sanadi, D. R. (Academic, New York), pp. 1. Meites, J. & Clemens, J. A. (1972) Vitam. Horm. (New York) 107-147. 30, 76-84. 18. Rudnick, G., Fishkes, M., Nelson, P. J. & Schuldiner, S. 2. MacLeod, R. M., Fontham, E. H. & Lehmeyer, J. E. (1970) (1980) J. Biol. Chem. 255, 3638-3641. Neuroendocrinology 6, 283-294. 19. Fujita, T., Kobayashi, S., Yui, R. & Iwanaga, T. (1980) in Hor- 3. Gibbs, D. M. & Neill, J. D. (1978) Endocrinology 102, 1895- mones, Adaptation and Evolution, ed. Ishii, S. (Springer, Ber- 1900. lin), pp. 35-43. 4. Caron, M. G., Beaulieu, M., Raymond, V., Gagne, B., 20. Vincent, J. D. & Dufy, B. (1982) in Cellular Regulation ofSe- Drouin, J., Lefkowitz, R. J. & Labrie, F. (1978) J. Biol. Chem. cretion and Release, ed. Conn, P. M. (Academic, New York), 253, 2244-2253. pp. 107-145. 5. Cronin, M. J., Roberts, J. M. & Weiner, R. I. (1978) Endocri- 21. Nansel, D. D., Gudelsky, G. A. & Porter, J. C. (1979) Endo- nology 103, 302-309. crinology 105, 1073-1077. 6. Cronin, M. J. & Weiner, R. I. (1979) Endocrinology 104, 307- 22. Gudelsky, G. A., Nansel, D. D. & Porter, J. C. (1980) Endo- '312. crinology 107, 30-34. Downloaded by guest on September 29, 2021