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Lovastatin is a potent inhibitor of cholecystokinin secretion in endocrine tumor cells in culture☆
Peptides 21 (2000) 553–557
Lovastatin is a potent inhibitor of cholecystokinin secretion in endocrine tumor cells in culture夞 Daesety Vishnuvardhan, Margery C. Beinfeld* Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA Received 7 December 1999; accepted 27 January 2000
Abstract Lovastatin prevents isoprene synthesis thereby affecting the structural organization of proteins involved in protein transport and secretion. Lovastatin at 1 M decreases CCK 8 secretion by over 50% in WE cells and in CCK 8 expressing AtT20 cells. At 10 M CCK 8 secretion was inhibited by two thirds and at 100 M, cytotoxic effects were observed in both cell types. Addition of mevalonate does not restore CCK secretion and stimulation of secretion by forskolin is also partially inhibited. Cellular content of CCK 8 and pro-CCK were not altered in either of these cell lines except at 100 M lovastatin. Our results clearly demonstrate that lovastatin at 1 M strongly inhibits CCK 8 secretion at multiple levels while having little or no effect on its synthesis. This effect on secretion may be partly responsible for the adverse gastrointestinal side effects of lovastatin in patients. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Lovastatin; Cholecystokinin; Prenylation
1. Introduction Lovastatin is a widely used drug for lowering serum cholesterol through inhibition of 3-hydroxy-3-methylglutaryl-coenzymeA (HMG CoA) reductase, the rate-limiting enzyme of the cholesterol biosynthetic pathway. It is an inactive lactone prodrug, which when enzymatically converted to its di-hydroxy open-acid form is a potent competitive inhibitor of liver HMG CoA reductase. It has also been used experimentally to define the role of isoprenylation in the function of a number of proteins. Prenylation is one of the most common post-translational lipid modifications of proteins and about 2% (by mass) of all cellular proteins may be modified in this way [17]. Post-translational prenylation of proteins in mammalian cells involves the formation of a thioether linkage between a 15-carbon farnesyl or a 20carbon gernylgeranyl moiety and one or more cysteine residues at or near the carboxyl terminus of the polypeptide. A few of the known farnesylated or geranylgeranylated proteins include nuclear lamin B [18], H- and K-Ras proteins 夞 This work was supported in part by NIH grants NS 18667 and NS 31602. * Corresponding author. Tel.: ⫹001-617-636-0346; fax: ⫹001-617636-6738. E-mail address: [email protected] (M.C. Beinfeld).
[20,13], the ␥-subunits of heterotrimeric G proteins [31,38], and Ras-related GTP binding proteins belonging to the Rab [25,27,28], Rac [26], Rap [12,24], Ral [26], and Rho [1,22] families. Protein isoprenylation also plays important role in the regulation of DNA synthesis and thus cell replication, and the in vitro addition of HMG-CoA reductase inhibitors have been observed to inhibit mitogen-stimulated lymphocyte proliferation [14,16,36]. When the HMG CoA reductase is inhibited by lovastatin in addition to its hypocholesterolemic effect, it is likely that it alters the biosynthesis of steroid hormones and may have multiple effects on nonsterol synthesis. Members of the family of Rab proteins are involved in various steps in the vesicular transport that functions between organelles of the endomembrane system [33]. Inhibition of isoprene synthesis by lovastatin leads to the cytosolic accumulation of unprenylated Rab proteins [23]. Rab GTPases represent a family of over 30 proteins that are localized to the surfaces of distinct membrane bound organelles. These proteins function in the process by which transport vesicles dock and/or fuse with their cognate, target membranes. Furthermore, the ␥-subunit of the heterotrimeric G proteins has been demonstrated to carry an isoprenoid modification [31,38]. Ras proteins are small GTPbinding proteins (G proteins) that play crucial roles in signaling pathways controlling cell growth and differentia-
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tion [10,11]. Ras proteins are farnesylated and the modification is required for oncogenic forms of Ras to transform cells [13,20,35]. The involvement of GTP-binding proteins in intracellular vesicular transport has been linked with several discrete steps in this process [34]. The most common adverse side effects of lovastatin treatment in humans are gastrointestinal disorders and lens opacities [29,37]. Based on the known effect of lovastatin on prenylation of proteins involved in intracellular vesicular transport, it was logical to infer that the gastrointestinal disorders may be caused, at least in part by inhibition of secretion of hormones and peptides. This study was initiated to examine the effect of lovastatin on secretion of Cholecystokinin (CCK) from two CCK expressing endocrine tumor cells in culture. Cholecystokinin is produced by both nerves and endocrine cells in the gut and by neurons. CCK is found in higher concentrations in the brain than in the gut, where it serves as a neurotransmitter or a neuromodulator in a number of important neuronal systems [5]. Like many hormones and neuropeptides, CCK is produced initially as a larger pre-prohormone. It passes through the regulated secretory pathway where it is processed to smaller bioactive forms and is secreted in response to specific stimuli [4].
2. Materials and methods 2.1. Cell culture and secretion experiments Rat medullary thyroid carcinoma cells (WE cells) were cultured as previously described [21] with the following modifications. They were plated in Minimum Essential Medium, Ham’s F-10 (1:1), 2.5% heat-inactivated donor calf serum and 5% horse serum containing 50 units/ml penicillin and 50 g/ml streptomycin (Penn-Strep). After 2 days, serum-free growth media was substituted for plating media, as described, except that it contained Penn-Strep. The cells were divided with trypsin–EDTA. Mouse pituitary tumor AtT20 cells were cultured in Dulbecco’s modified Eagle’s Minimal Essential Medium (DMEM) containing 10% newborn calf serum, 10% horse serum, and Penn-strep. AtT20 cells were transfected using calcium phosphate with pcDNA3 containing the rat CCK cDNA. Stably transfected cells were selected with Geneticin (G418). The cell line selected for this study was called AE7 [8]. For the release experiments, the cells were plated into 6 well plates and were used about a week after plating. Cell culture components were from Life Technologies, Inc. (Rockville, MD, USA). Lovastatin was from Calbiochem (La Jolla, CA, USA) and was dissolved in DMSO. Lovastatin to be tested was added to normal growth medium, with 1 ml/well and incubated for 2 days. Mevalonic acid or forskolin (Sigma Chemical Co., St. Louis, MO, USA) was added along with lovastatin. When the CCK and pro-CCK content of cells was measured, the cells were removed from the wells by scraping in the presence of 1 ml of 0.1 M HCl, sonicated,
Fig. 1. Effect of lovastatin on secretion of (A) CCK 8 in WE cells, (B) CCK 8 in AE7 cells. *P ⬍ 0.05.
and centrifuged and an aliquot was removed and neutralized with 0.1 M NaOH before assay. Amidated CCK and proCCK immunoreactivity was assayed by radioimmunoassay as described previously [7,2]. When comparisons were made to control untreated cells, one-way ANOVA on transformed ranks was used with Bonferroni t-test.
3. Results Two CCK expressing cell lines WE and AE7 were used to study the effect of lovastatin on CCK secretion. Both cells process pro-CCK completely to CCK 8 amide, which they secrete. Both contain and also secrete intact, nonprocessed pro-CCK. Lovastatin at 1 M decreased the CCK 8 secretion more than 50% in WE cells and at 10 M more than 90% was inhibited (Fig. 1A). In the presence of 100 M of lovastatin CCK 8 secretion was barely detectable (Fig. 1A). In AE7 cells, lovastatin at 1 M inhibited CCK 8 secretion about 50% and at 10 M and 100 M, CCK 8 secretion was inhibited by over 70% (Fig. 1B). Lovastatin at 100 M was very toxic to both cell lines, causing them to round up and come off the plates. This effect was more pronounced with WE cells than with AE7 cells. Simultaneous addition of mevalonate at 10 M or at 30 M to the culture medium does not restore the secretion (Fig. 2). Since CCK secretion is regulated by intracellular cAMP level in these cell lines [3,6], the effect of forskolin on the inhibition of secretion was studied. As shown in Fig. 2G, the results imply that lovastatin alters the stimulatory effect of forskolin on secretion. In order to further evaluate the effect of lovastatin on CCK biosynthesis, cellular levels of pro-CCK, and amidated CCK 8 were determined. Cellular levels of amidated
D. Vishnuvardhan, M.C. Beinfeld / Peptides 21 (2000) 553–557
Fig. 2. Effect of mevalonate and forskolin on the inhibition of CCK 8 secretion by lovastatin in WE cells. A, control; B, 1 M lovastatin; C, 1 M lovastatin ⫹ 10 M mevalonate; D, 10 M mevalonate; E, 1 M lovastatin ⫹ 30 M mevalonate. F, 30 M mevalonate; G, 1 M lovastatin ⫹ 10 M forskolin; H, 10 M forskolin. *P ⬍ 0.001; **P ⬍ 0.007; ***P ⬍ 0.002 compared to control untreated cells.
CCK 8 were not significantly altered even at 10 M lovastatin (Fig. 3A,B) where a very strong inhibition of CCK 8 secretion was seen. This was true in both the cell lines. Cellular levels of pro-CCK were also not affected even at 10 M lovastatin (Fig. 3C,D).
4. Discussion Lovastatin at 1 M has a profound inhibitory effect on secretion of amidated CCK 8. This inhibition could be
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caused by cholesterol depletion even though ER has low cholesterol content as compared with the plasma membrane. Early work by Orci et al. [32] suggested that a gradient of cholesterol exists across the Golgi stack. Budding vesicles selectively enriching their membrane with cholesterol would maintain this gradient. These vesicles would, therefore, have a thicker membrane than the compartment from which they bud. In the absence of replenishing amounts of cholesterol the membrane and vesicle structure may disorganize decreasing secretion. The profound effect of lovastatin on CCK 8 secretion could also be due to inhibition of prenylation of multiple proteins involved in vesicle transport and secretion. Lovastatin has additional effects at 30 –50 M including disassembly of actin stress fibers [19], inhibition of protein synthesis [9] and induction of apoptosis [30]. In this dose range, lovastatin is probably toxic to the WE and AE7 cells and in this study toxicity was observed at 100 M. Lovastatin effect on secretion at 1 M is not accompanied by any decrease in CCK 8 or pro-CCK content, so it is unlikely to be caused solely by inhibition of protein synthesis. Depletion of mevalonic acid, obtained by inhibition of HMG CoA reductase by lovastatin affects several cellular processes. Mevalonate is essential for cell proliferation and survival because it is a precursor of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, the isoprenyl donors involved in the posttranslational modification of key regula-
Fig. 3. Effect of lovastatin on the cellular content of (A) CCK 8 in WE cells, (B) CCK 8 in AE7 cells, (C) pro-CCK in WE cells and (D) pro-CCK in AE7 cells. *P ⬍ 0.05.
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tory proteins. Several studies have shown that the effects of lovastatin can be prevented by simultaneous addition of mevalonate [15,30]. Unexpectedly, replenishment of mevalonate does not bypass the metabolic block imposed by lovastatin. Since lovastatin was known to decrease adenylyl cyclase activity [15], it is also possible that lovastatin inhibitory effect on CCK 8 secretion was through the decrease in cAMP production. The addition of lovastatin partially inhibits the response to the cAMP activator forskolin. Our study clearlys demonstrate that lovastatin strongly inhibited CCK 8 secretion at concentrations which have little effect on CCK synthesis and may affect the secretory pathway at multiple levels. Very similar results were obtained with two very different CCK expressing cells, one a naturally expressing rat medullary thyroid carcinoma cell and the other an engineered mouse pituitary tumor cell line whose expression of CCK is driven by the CMV promoter. This inhibition of secretion is likely to occur with other peptides and hormones and may contribute to gastrointestinal disorders in patients treated with this drug.
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[20]
[21]
References [1] Adamson P, Marshall CJ, Hall A, Tilbrook PA. Post-translational modifications of p21rho proteins. J Biol Chem 1992;267:20033– 8. [2] Beinfeld MC. Cholecystokinin (CCK) gene-related peptides: distribution and characterization of immunoreactive pro-CCK and an amino-terminal pro-CCK fragment in rat brain. Brain Res 1985;344: 351–5. [3] Beinfeld MC. CCK mRNA expression, pro-CCK processing and regulated secretion of immunoreactive CCK peptides by rat insulinoma cells (RIN 5F) and mouse pituitary tumor (AtT20) cells in culture. Neuropeptides 1992;22:213–7. [4] Beinfeld MC. CCK biosynthesis and processing: recent progress and future challenges. Life Sci 1997;61:2359 – 66. [5] Beinfeld MC. Cholecystokinin/gastrin. In. Watson JS, editor. Psychopharmacology, chapt. 56, CD/ROM ed. Philadelphia: LippincottRaven Press, 1998. [6] Beinfeld MC, Haun RS, Allard LR, Dixon JE. Regulation of cholecystokinin secretion from a rat thyroid medullary carcinoma cell line: role of calcium, cyclic nucleotides, glucocorticoids, neurotensin, and calcitonin gene-related peptide. Peptides 1992;13:545–90. [7] Beinfeld MC, Meyer DK, Eskay RL, Jensen RT, Brownstein MJ. The distribution of cholecystokinin immunoreactivity in the central nervous system of the rat as determined by radioimmunoassay. Brain Res 1981;212:51–7. [8] Beinfeld MC, Perloff MD, Venkatakrishnan K. Identification of glycine-extended CCK peptides in endocrine cells and modulation of CCK amide and CCK Gly content and secretion from endocrine tumor cells by an inhibitor of amidation. Peptides 1998;19:1393– 8. [9] Bhullar RP. Expression of a 24 kDa GTP-binding protein (Gn24) is increased in lovastatin treated human erythroleukemia cells. Mol Cell Biochem 1996;156:59 – 67. [10] Boguski MS, McCormick F. Proteins regulating Ras and its relatives. Nature 1993;366:643–54. [11] Burgering BM, Bos JL. Regulation of Ras-mediated signalling: more than one way to skin a cat. Trends Biochem Sci 1995;20:18 –22. [12] Buss JE, Quilliam LA, Kato K, Casey PJ, Solski PA, Wong G, Clark R, McCormick F, Bokoch GM, Der CJ. The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports trans-
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D. Vishnuvardhan, M.C. Beinfeld / Peptides 21 (2000) 553–557 plexes across the cisternae of the Golgi apparatus. Proc Natl Acad Sci USA 1981;78:293–7. [33] Pfeffer SR. Rab GTPases: master regulators of membrane trafficking. Curr Opin Cell Biol 1994;6:522– 6. [34] Rothman JE. Mechanisms of intracellular protein transport. Nature 1994;372:55– 63. [35] Schafer WR, Kim R, Sterne R, Thorner J, Kim SH, Rine J. Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans. Science 1989;245:379 – 85.
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Lovastatin is a potent inhibitor of cholecystokinin secretion in endocrine tumor cells in culture夞 Daesety Vishnuvardhan, Margery C. Beinfeld* Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA Received 7 December 1999; accepted 27 January 2000
Abstract Lovastatin prevents isoprene synthesis thereby affecting the structural organization of proteins involved in protein transport and secretion. Lovastatin at 1 M decreases CCK 8 secretion by over 50% in WE cells and in CCK 8 expressing AtT20 cells. At 10 M CCK 8 secretion was inhibited by two thirds and at 100 M, cytotoxic effects were observed in both cell types. Addition of mevalonate does not restore CCK secretion and stimulation of secretion by forskolin is also partially inhibited. Cellular content of CCK 8 and pro-CCK were not altered in either of these cell lines except at 100 M lovastatin. Our results clearly demonstrate that lovastatin at 1 M strongly inhibits CCK 8 secretion at multiple levels while having little or no effect on its synthesis. This effect on secretion may be partly responsible for the adverse gastrointestinal side effects of lovastatin in patients. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Lovastatin; Cholecystokinin; Prenylation
1. Introduction Lovastatin is a widely used drug for lowering serum cholesterol through inhibition of 3-hydroxy-3-methylglutaryl-coenzymeA (HMG CoA) reductase, the rate-limiting enzyme of the cholesterol biosynthetic pathway. It is an inactive lactone prodrug, which when enzymatically converted to its di-hydroxy open-acid form is a potent competitive inhibitor of liver HMG CoA reductase. It has also been used experimentally to define the role of isoprenylation in the function of a number of proteins. Prenylation is one of the most common post-translational lipid modifications of proteins and about 2% (by mass) of all cellular proteins may be modified in this way [17]. Post-translational prenylation of proteins in mammalian cells involves the formation of a thioether linkage between a 15-carbon farnesyl or a 20carbon gernylgeranyl moiety and one or more cysteine residues at or near the carboxyl terminus of the polypeptide. A few of the known farnesylated or geranylgeranylated proteins include nuclear lamin B [18], H- and K-Ras proteins 夞 This work was supported in part by NIH grants NS 18667 and NS 31602. * Corresponding author. Tel.: ⫹001-617-636-0346; fax: ⫹001-617636-6738. E-mail address: [email protected] (M.C. Beinfeld).
[20,13], the ␥-subunits of heterotrimeric G proteins [31,38], and Ras-related GTP binding proteins belonging to the Rab [25,27,28], Rac [26], Rap [12,24], Ral [26], and Rho [1,22] families. Protein isoprenylation also plays important role in the regulation of DNA synthesis and thus cell replication, and the in vitro addition of HMG-CoA reductase inhibitors have been observed to inhibit mitogen-stimulated lymphocyte proliferation [14,16,36]. When the HMG CoA reductase is inhibited by lovastatin in addition to its hypocholesterolemic effect, it is likely that it alters the biosynthesis of steroid hormones and may have multiple effects on nonsterol synthesis. Members of the family of Rab proteins are involved in various steps in the vesicular transport that functions between organelles of the endomembrane system [33]. Inhibition of isoprene synthesis by lovastatin leads to the cytosolic accumulation of unprenylated Rab proteins [23]. Rab GTPases represent a family of over 30 proteins that are localized to the surfaces of distinct membrane bound organelles. These proteins function in the process by which transport vesicles dock and/or fuse with their cognate, target membranes. Furthermore, the ␥-subunit of the heterotrimeric G proteins has been demonstrated to carry an isoprenoid modification [31,38]. Ras proteins are small GTPbinding proteins (G proteins) that play crucial roles in signaling pathways controlling cell growth and differentia-
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tion [10,11]. Ras proteins are farnesylated and the modification is required for oncogenic forms of Ras to transform cells [13,20,35]. The involvement of GTP-binding proteins in intracellular vesicular transport has been linked with several discrete steps in this process [34]. The most common adverse side effects of lovastatin treatment in humans are gastrointestinal disorders and lens opacities [29,37]. Based on the known effect of lovastatin on prenylation of proteins involved in intracellular vesicular transport, it was logical to infer that the gastrointestinal disorders may be caused, at least in part by inhibition of secretion of hormones and peptides. This study was initiated to examine the effect of lovastatin on secretion of Cholecystokinin (CCK) from two CCK expressing endocrine tumor cells in culture. Cholecystokinin is produced by both nerves and endocrine cells in the gut and by neurons. CCK is found in higher concentrations in the brain than in the gut, where it serves as a neurotransmitter or a neuromodulator in a number of important neuronal systems [5]. Like many hormones and neuropeptides, CCK is produced initially as a larger pre-prohormone. It passes through the regulated secretory pathway where it is processed to smaller bioactive forms and is secreted in response to specific stimuli [4].
2. Materials and methods 2.1. Cell culture and secretion experiments Rat medullary thyroid carcinoma cells (WE cells) were cultured as previously described [21] with the following modifications. They were plated in Minimum Essential Medium, Ham’s F-10 (1:1), 2.5% heat-inactivated donor calf serum and 5% horse serum containing 50 units/ml penicillin and 50 g/ml streptomycin (Penn-Strep). After 2 days, serum-free growth media was substituted for plating media, as described, except that it contained Penn-Strep. The cells were divided with trypsin–EDTA. Mouse pituitary tumor AtT20 cells were cultured in Dulbecco’s modified Eagle’s Minimal Essential Medium (DMEM) containing 10% newborn calf serum, 10% horse serum, and Penn-strep. AtT20 cells were transfected using calcium phosphate with pcDNA3 containing the rat CCK cDNA. Stably transfected cells were selected with Geneticin (G418). The cell line selected for this study was called AE7 [8]. For the release experiments, the cells were plated into 6 well plates and were used about a week after plating. Cell culture components were from Life Technologies, Inc. (Rockville, MD, USA). Lovastatin was from Calbiochem (La Jolla, CA, USA) and was dissolved in DMSO. Lovastatin to be tested was added to normal growth medium, with 1 ml/well and incubated for 2 days. Mevalonic acid or forskolin (Sigma Chemical Co., St. Louis, MO, USA) was added along with lovastatin. When the CCK and pro-CCK content of cells was measured, the cells were removed from the wells by scraping in the presence of 1 ml of 0.1 M HCl, sonicated,
Fig. 1. Effect of lovastatin on secretion of (A) CCK 8 in WE cells, (B) CCK 8 in AE7 cells. *P ⬍ 0.05.
and centrifuged and an aliquot was removed and neutralized with 0.1 M NaOH before assay. Amidated CCK and proCCK immunoreactivity was assayed by radioimmunoassay as described previously [7,2]. When comparisons were made to control untreated cells, one-way ANOVA on transformed ranks was used with Bonferroni t-test.
3. Results Two CCK expressing cell lines WE and AE7 were used to study the effect of lovastatin on CCK secretion. Both cells process pro-CCK completely to CCK 8 amide, which they secrete. Both contain and also secrete intact, nonprocessed pro-CCK. Lovastatin at 1 M decreased the CCK 8 secretion more than 50% in WE cells and at 10 M more than 90% was inhibited (Fig. 1A). In the presence of 100 M of lovastatin CCK 8 secretion was barely detectable (Fig. 1A). In AE7 cells, lovastatin at 1 M inhibited CCK 8 secretion about 50% and at 10 M and 100 M, CCK 8 secretion was inhibited by over 70% (Fig. 1B). Lovastatin at 100 M was very toxic to both cell lines, causing them to round up and come off the plates. This effect was more pronounced with WE cells than with AE7 cells. Simultaneous addition of mevalonate at 10 M or at 30 M to the culture medium does not restore the secretion (Fig. 2). Since CCK secretion is regulated by intracellular cAMP level in these cell lines [3,6], the effect of forskolin on the inhibition of secretion was studied. As shown in Fig. 2G, the results imply that lovastatin alters the stimulatory effect of forskolin on secretion. In order to further evaluate the effect of lovastatin on CCK biosynthesis, cellular levels of pro-CCK, and amidated CCK 8 were determined. Cellular levels of amidated
D. Vishnuvardhan, M.C. Beinfeld / Peptides 21 (2000) 553–557
Fig. 2. Effect of mevalonate and forskolin on the inhibition of CCK 8 secretion by lovastatin in WE cells. A, control; B, 1 M lovastatin; C, 1 M lovastatin ⫹ 10 M mevalonate; D, 10 M mevalonate; E, 1 M lovastatin ⫹ 30 M mevalonate. F, 30 M mevalonate; G, 1 M lovastatin ⫹ 10 M forskolin; H, 10 M forskolin. *P ⬍ 0.001; **P ⬍ 0.007; ***P ⬍ 0.002 compared to control untreated cells.
CCK 8 were not significantly altered even at 10 M lovastatin (Fig. 3A,B) where a very strong inhibition of CCK 8 secretion was seen. This was true in both the cell lines. Cellular levels of pro-CCK were also not affected even at 10 M lovastatin (Fig. 3C,D).
4. Discussion Lovastatin at 1 M has a profound inhibitory effect on secretion of amidated CCK 8. This inhibition could be
555
caused by cholesterol depletion even though ER has low cholesterol content as compared with the plasma membrane. Early work by Orci et al. [32] suggested that a gradient of cholesterol exists across the Golgi stack. Budding vesicles selectively enriching their membrane with cholesterol would maintain this gradient. These vesicles would, therefore, have a thicker membrane than the compartment from which they bud. In the absence of replenishing amounts of cholesterol the membrane and vesicle structure may disorganize decreasing secretion. The profound effect of lovastatin on CCK 8 secretion could also be due to inhibition of prenylation of multiple proteins involved in vesicle transport and secretion. Lovastatin has additional effects at 30 –50 M including disassembly of actin stress fibers [19], inhibition of protein synthesis [9] and induction of apoptosis [30]. In this dose range, lovastatin is probably toxic to the WE and AE7 cells and in this study toxicity was observed at 100 M. Lovastatin effect on secretion at 1 M is not accompanied by any decrease in CCK 8 or pro-CCK content, so it is unlikely to be caused solely by inhibition of protein synthesis. Depletion of mevalonic acid, obtained by inhibition of HMG CoA reductase by lovastatin affects several cellular processes. Mevalonate is essential for cell proliferation and survival because it is a precursor of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, the isoprenyl donors involved in the posttranslational modification of key regula-
Fig. 3. Effect of lovastatin on the cellular content of (A) CCK 8 in WE cells, (B) CCK 8 in AE7 cells, (C) pro-CCK in WE cells and (D) pro-CCK in AE7 cells. *P ⬍ 0.05.
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tory proteins. Several studies have shown that the effects of lovastatin can be prevented by simultaneous addition of mevalonate [15,30]. Unexpectedly, replenishment of mevalonate does not bypass the metabolic block imposed by lovastatin. Since lovastatin was known to decrease adenylyl cyclase activity [15], it is also possible that lovastatin inhibitory effect on CCK 8 secretion was through the decrease in cAMP production. The addition of lovastatin partially inhibits the response to the cAMP activator forskolin. Our study clearlys demonstrate that lovastatin strongly inhibited CCK 8 secretion at concentrations which have little effect on CCK synthesis and may affect the secretory pathway at multiple levels. Very similar results were obtained with two very different CCK expressing cells, one a naturally expressing rat medullary thyroid carcinoma cell and the other an engineered mouse pituitary tumor cell line whose expression of CCK is driven by the CMV promoter. This inhibition of secretion is likely to occur with other peptides and hormones and may contribute to gastrointestinal disorders in patients treated with this drug.
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