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Calcium requirements for barium stimulation of enkephalin and vasoactive intestinal peptide biosynthesis in adrenomedullary chromaffin cells
h’europepddes (1988) 11.39-45 0 Longman GroupUK Ltd 1988
Calcium Requirements for Barium Stimulation of Enkephalin and Vasoactive Intestinal Peptide Biosynthesis in Adrenomedullary Chromaffin CellIs JAMES A. WASCHEK
and LEE E. ElDEN
Unit on Molecular and Cellular Neurobiology, Health, National Institutes of Health; Building J. W.1
Laboratory of Cell Biology, National institute of Mental 36, Room 3A-17; Bethesda, MD 20892 (reprint requests to
Abstract-The divalent cation barium was used to study the role of calcium in coupling neuropeptide secretion and biosynthesis following secretagogue stimulation of bovine chromaffin cells. Barium chloride (0.1-2.5 mM) stimulated in a dose-dependent manner the secretion of met-enkephalin (up to 20% of intracellular peptide content) and increased the total amount (cell plus medium content) of met-enkephalin and vasoactive intestinal polypeptide (VIP) 2- to 3-fold after 72 hours. A greater than six-fold increase in proenkephalin mRNA (mRNAenk) was observed by 24 hours following barium stimulation. The voltagesensitive calcium channel blocker D600 inhibited the barium-stimulated secretion of enkephalin and blocked the stimulation of VIP biosynthesis and mRNAenk. Reducing calcium in the medium resulted in an enhancement of barium-stimulated release of both peptides, but blocked the induction of their biosynthesis. The data indicate that calcium targets involved in secretion can be activated by barium or calcium while calcium targets involved in biosynthesis specifically require calcium. It is therefore proposed that pathways leading to peptide secretion and biosynthesis in the adrenal diverge just after secretagogue-stimulated calcium influx.
Introduction
intestinal polypeptide (VIP), by either nicotinic stimulation or depolarization, is accompanied by an increase in peptide biosynthesis, which occurs at a pretranslational locus (4-6). The secretion and enhanced biosynthesis of met-enkephalin and VIP can be inhibited by reducing extracellular calcium or by treatment with agents that block influx of calcium into the cell (3,5-7). The term stimulussecretion-synthesis coupling has been applied to the coordinate calcium-dependent stimulation of both neuropeptide secretion and biosynthesis by
Stimulation of chromaffin cells either with the secretogogue acetylcholine or by depolarization with elevated potassium causes catecholamines and secretory peptides to be released by exocytosis from the vesicles in which they are stored into the extracellular space (l-3). Release of at least two of these peptides, met-enkephalin and vasoactive Date received 16 September 1987 Date accepted 21 September 1987
39
40 chromaffin cell-specific secretagogues (4,7). In the present study, the sites of calcium action within the chromaffin cell are examined using the divalent cation barium, which has been previously used to study secretion in the adrenal medulla and other neuroendocrine tissues (8-14). Barium apparently depolarizes the chromaffin cell (15)) presumably because of its ability to penetrate voltagedependent calcium channels and its ability to block calcium-dependent outwardly-directed potassium current (16). The results presented here indicate that calcium targets for peptide secretion vs. biosynthesis are markedly different in their sensitivity to barium. Materials and Methods Cell Culture. Chromaffin cells were obtained from fresh bovine adrenal medulla and were cultured in a humidified 95% sir/5% carbon dioxide atmosphere as described (3) in non-collagen coated Coastar 24-well dishes (16-mm diameter) at a density of 750000 to 1000000 cells per well in the Basal Medium of Eagle with Earle’s salts and HEPES, 25 mM, supplemented with 5% fetal bovine serum (Hyclone), penicillin 100 units/ml, streptomycin 100 @ml, and cytosine arabinoside 1OpM. After 2 to 3 days in culture, 30 to 60 minutes before adding drugs, medium was removed and replaced with fresh medium diluted 1:l with a solution of chloride salts of sodium, potassium, calcium, and magnesium, at concentrations of 154, 5.4, 1.8, and 0.8 mM, respectively. Replacement of sulfate, carbonate and phosphate salts with the chloride salts in this manner prevented precipitation of barium salts, which occurred in the standard culture medium at barium concentrations greater than 2 mM after 4 to 5 days. The preincubation medium contained D600 (Knoll) at doses indicated or reduced calcium in experiments where barium was to be given under these conditions. In one set of experiments, where bariuminduced met-enkephalin release was measured over several calcium concentrations, a special medium (SRM (3)) which contained no fetal bovine serum (and therefore no added calcium from either supplemental salts or serum) was used for preincubation and measurement of peptide release. Immediately before experiments, preincubation medium was removed and replaced with identical medium containing various concentrations of barium. Release experiments were carried out over a 30-minute time interval unless otherwise specified. Biosynthesis experiments
NEUKOPEPTIDES
were carried out for 24 hours (mRNA levels) and 72 hours (peptide levels). Peptide radioimmunoassay.
Met-enkephalin and VIP immunoreactivity in medium and cell extracts were measured as described (3) using metenkephalin antiserum (RB-4) kindly provided by Dr. Steve Sabol (NHLBI, National Institutes of Health) and VIP antiserum N12 kindly provided by Dr.. Gajanan Nilaver, Columbia University College of Physicians and Surgeons (17). mRNAenk quantitation. RNA was extracted from cells, denatured, and electrophoresed on agarose gels as described (18). Total RNA extracted was estimated by densitometric scanning of the 28s band appearing on photographic negatives of UV illuminated gels stained with ethidium bromide. The RNA was then transblotted from the gel to Gene Screen (NEN) and hybridized with a nicktranslated 400 base-pair Pst I fragment of the cDNA encoding bovine preproenkephalin A (19). mRNAenk was quantitifed by densitometric scanning of film exposed to the hybridized blots. In some cases, after total RNA measurement, mRNAenk was quantified by direct slot blot RNA application because hybridization to nonmRNAenk species was negligible.
Results Release of met-enkephalin by barium chloride was dose dependent and was maximal (22% of cell content) at 4 mM (Fig. 1). To determine whether or not barium acted directly at calcium sites or indirectly by facilitating the action of calcium, cells were treated with barium, 1 mM and 2.5 mM, in the presence of normal (1.8 mM) and reduced (0.1 mM) concentrations of extracellular calcium and in the presence of D600, which blocks voltagedependent calcium channels. Release at both barium concentrations was enhanced in medium with reduced calcium, and was inhibited by D600 (Fig. 2). Release of met-enkephalin from cells treated with 2 mM barium over a range of calcium concentrations was maximal in the absence of added calcium and was reduced several-fold at a calcium concentration of 10 mM (Fig. 3). The release of VIP elicited by 2 mM barium was likewise inversely related to the extracellular calcium concentration, and was maximal in the total absence of added extracellular calcium (Table 1). Thus, barium probably enters the cell through the D600-sensitive calcium channel and
CALCIUM
REQUIREMENTS
FOR BARIUM
STIMULATION
-11
OF ENKEPHALIN
Table 1 Inhibation of Barium-Stimulated Release of Vasoactive Intestinal Polypeptide (VIP) in Bovine Chromaffin Cells by Extracellular Calcium Concentration of CaCl, added
ControI BaCI,, 2mM
OmM
2.2 mM
20.5 f 2.7 99.7 * 6.6
23.5 + 5.6 59.3 + 2.5
10mM 16.3 i 10.1 39.2 + 3.2
Cells were preincubated for 1 hr at 37” in serum-free medium containing the indicated concentration of added calcium chloride. Concentration of VIP in the medium was measured 1 hr after addition of barium (values are pg/well. mean f SEM. n= 3).
253
20,
Fig 2 Barium-stimulated release of enkephalin by bovine adrenal chromaffin cells over 30 minutes (% of cell contents ? SEM. n=3) in normal (N) medium (1.8 mM Ca”), in low calcium (LC) medium (100 uM). and in normal medium containing the calcium channel blocker, D600, 10 PM. Cells were preincubated for 1 hr in the indicated medium before addition of 1 mM barium. Mean total met-enkephaiin content/ well (cells plus medium) was 2.7 to 2.9 ng. T = p< .05 compared with respective value in control group. 1 = p<.O5 compared with barium control (Student’s t-test).
3 E ae 15r
0
70 -
1.0
2.0
3.0
4.0 60-
Barium Cont., mM Fig 1 Release affin cells over of barium (% met-enkephalin “g.
of met-enkephalin by bovine adrenal chrom30 minutes after stimulation with several doses of cell contents, 2 SEM, n=3). Mean total content/well (cells plus medium) was 3.7 to 4.1
causes met-enkephalin
and VIP release by acting directly at intracellular calcium sites. Met-enkephalin and VIP levels (cells plus medium) increased with time following treatment with 2.5 mM barium; increases in both peptides were fully apparent after 72 hours (Fig. 4). Maximal elevation of both peptides at 72 hours was observed with 1 mM barium (Fig. 5). Induction of met-enkephalin peptide by barium occurred in association with increased levels of mRNAenk measured at 24 hours (Fig. 6), in agreement with a recent report (20). Thus, barium appears to stimulate met-enkephalin biosynthesis at a pretranslational locus, as do nicotine and depolarizing concentrations of potassium ion (4,5). Barium induction of met-enkephalin and VIP peptide and mRNAenk (Fig. 7) was inhibited by reducing extracellular calcium. This was in con-
504O3020 10 0.
’
No calcium
// N 5.0
-log
I
I
4.0
3.0
-
9 2.0
[Ca2+I, M added
Fig 3 Release of met-enkephalin by bovine adrenal chromaftin cells over 60 minutes 1% of cell contents + SEM, n=3) at several concentrations of extracellular calcium when treated with (0) or without (0) barium, 2 mM. Mean total metenkephalin content/well (cells plus medium) was 2.0 to 2.7 ng.
trast to the enhancement of barium-stimulated secretion caused by this treatment, and suggest that calcium targets for these processes are different. Induction of VIP, but not metenkephalin peptide or mRNAenk by barium was blocked by 10 PM D600 (Table 2). D600 did
NEUROPEPTIDES
12r
0.0
C
Ba
C
15 hr
Ba
40 hr
C
Ba
72 hr
Fig 4 Time-course in increase in cell and medium VIP (upper frame) and met-enkephalin (lower frame) after treatment of bovine chromaffin cells with barium, 2.5 mM over 72 hours. Peptide contents measured in the cell and medium are indicated by shaded and open bars, respectively. SEM values are for cell peptide and total peptide contents (n=3).
Table 2
Effect
Proenkephalin
Control(d) Barium 1 mM Barium 1 mM +D600 10 uM Barium 1 mM +D600 30 PM
0.1
0.2
0.5
Barium concentration,
1.0
2.0
mM
Fig 5 Total (cell and medium) VIP (upper frame) and met-enkephalin (lower frame) 72 hours after treatment of bovine chromaftin cells with several doses of barium. Peptide contents measured in the cell and medium are indicated by shaded and open bars, respectively. SEM values (n=3) are for cell peptide and total peptide contents. 1 = p<.O5 compared with untreated control value (Student’s t-test).
of D600 on 1 mM Barium Induction of Met-Enkephalin and VIP Levels after 72 Hours and mRNA after 24 Hours Treatment of Bovine Chromaffin Cells(a) VIP(b)
met-enkephalin(b)
51.8 f 1.5 121.4 + 2.7(Y) 40.3 zb 3.3(Y,l)
3618 f 357 10439 f 387(Y) 10199 + 361(Y)
Proenkephalin
1.0 + 0.1 4.2 + 0.5(Y) 4.3 f 0.3(V)
mRNA(c)
n.d.
nd.
2.0 f 0.3(Y.1)
(a) Values are mean + SEM, n=3 for all treatments except barium 1 mM alone (n=9) (b) Total contents in cells and medium, pg/well. (c) Standarized to total RNA extracted and normalized to control value. Data are pooled from 3 separate experiments, n=3 to 9. (d) Control values of D600 were not significantly lower than untreated controlvalues. (Y) pc.05 compared with control group. (7) p-z.05 compared with barium alone group. (n.d.) not determined
CALCIUM
REQUIREMENTS
FOR BARIUM
STIMULATION
43
OF ENKEPHALIN
Barium, 1 mM
Y
120 80 - Barium, 0 mM 40.
01
’
0.0
1
1
I
1.0
2.0
3.0
1
I
4.0
O-
N LC
N LC
N LC
N LC
Barium Concentration, mM Fig 6 Concentration of mRNA’“’ in bovine chromaffin cells (relative to control) measured 24 hours after treatment with several doses of barium. Individual values (fSEM, n=3) are corrected for total RNA extracted, as described in methods.
inhibit the barium-stimulated increase in mRNAenk at 30 PM, a dose higher than that needed to inhibit barium stimulated metenkephalin secretion (Fig. 2), or to inhibit induction of met-enkephalin peptide and mRNA by nicotine or depolarizing concentrations of potassium (4,6,7). Discussion The data presented here demonstrate that barium stimulates both release and biosynthesis of neuropeptides in chromaffin cells, consistent with previous studies on secretion in bovine chromaffin cells (8,11-14), and with a recent report showing enkephalin mRNA induction by barium (20). The data here further show that biosynthesis of VIP can also be stimulated by barium, and that barium cannot fully substitute for calcium in stimulation of biosynthesis of either peptide. Thus, barium stimulates biosynthesis of these peptides indirectly, presumably by depolarizing the cell and allowing calcium to enter the cell to act at the appropriate intracellular sites. Secretion, on the other hand, was elicited in the complete absence of added extracellular calcium, indicating that barium probably acted directly at calcium sites to stimulate secretion. It could be argued that barium transiently increased intracellular calcium by displacing it from intracellular stores to cause secretion. However, a corresponding effect on biosynthesis was not observed. Additional support for a direct agonist action of barium in secretion is that barium is able to produce caIcium-independent secretion from permeabilized chromaffin cells (21). In any
12 8 4 0
6-
Barium 1.0 mM Y
Barium, 0 mM
N LC
N LC
Fig 7 Effect of medium containing reduced calcium on the barium-stimulated biosynthesis of VIP and met-enkephalin in bovine chromaffin cells. Total peptide levels (cell and medium) of VIP (upper) and met-enkephalin (middle) after 72 hours treatment and proenkephalin mRNA levels (lower) after 24 hours treatment with 1 mM barium in normal (N) medium (1.8 mM Ca2+), or in low calcium (LC) medium (100 uM CaZ+). Peptide contents measured in the cell and medium in upper two panels are indicated by darkened and open bars, respectively, with SEM values referring to cell and total peptide contents. Proenkephalin mRNA values (lower panel), corrected for total RNA extracted as described in the methods, are normalized to control values. N = 3 for all peptides experiments; n = 6 to 9 for RNA experiments (pooled data from 2 experiments). f = p<.O5 compared with respective value in control group. 7 = p<.O5 compared with barium control value (Student’s t-test).
44
NEUROPEPTIDES
event, it is shown here that barium distinguishes calcium-dependent processes involved in secretion versus biosynthesis of met-enkephalin in chromaffin cells. Biosynthesis and release of another peptide, VIP, was also stimulated by barium. Induction of VIP biosynthesis by barium was more sensitive to D600 than was induction of enkephalin. VIP appears to be inducible in a small subset of cells distinct from those that express met-enkephalin (22). This differential regulation thus appears to be exhibited in two different cell types, and may reflect differences at the cell membrane or at intracellular sites. Induction of VIP by nicotine does appear to be at the level of mRNA induction because increased peptide levels are blocked by cycloheximide (6). A direct comparison of effects on met-enkephalin and VIP mRNA could not be made because a polynucleotide probe to measure bovine VIP mRNA is not currently available. Cyclic AMP as well as calcium may be involved in the biosynthesis of met-enkephalin and VIP in
chromaffin cells (23). Since nicotine stimulation of met-enkephalin biosynthesis is calcium dependent, and nicotine causes an increase in intracellular CAMP (4,7,23), it is possible that calcium and CAMP may act cooperatively to stimulate biosynthesis or that calcium acts via an increase in CAMP. An additional possibility is that CAMP is important in regulating enkephahn and VIP in neuroendocrine cells during expression development, but is not required for stimulussecretion-synthesis coupling in the mature chromaffin cell. The data presented here and in the literature are consistent with the model for the role of calcium in secretagogue-stimulated peptide release and the compensatory increase in peptide biosynthesis Calcium voltage-sensitive calcium channels. enters the cell and activates factors associated with secretion and other distinct factors associated with regulation of peptide biosynthesis. Since barium appears to bind calmodulin poorly and activates some calmodulin-mediated processes weakly if at
Paptide
biosynthesis
Ca*+ 0 Ba** n
Peptide
secretion
Fig 8 Model depicting the interaction of calcium and barium with regulatory factors involved in peptide secretion and the compensatory increase in biosynthesis of secreted peptides in bovine chromaffin cells. Following stimulation with acetylcholine or by depolarization, calcium enters the cell through voltage-sensitive calcium channels and stimulates peptide secretion and biosynthesis by interacting at the distinct calcium binding sites that govern these processes. Barium stimulates peptide release by directly binding and activating calcium targets associated with secretion, but stimulates biosynthesis only by facilitating the action of calcium. Barium may facilitate the action of calcium by depolarizing the cell and allowing calcium entry into the cell through voltage-dependent calcium channels.
CALCIUM
REQUIREMENTS
FOR BARIUM
STIMULATION
OF ENKEPHALIN
all (24-26), the data here argue against an important regulatory role for calmodulin in chromaffin cell secretion, but are not inconsistent with a role of calmodulin in peptide biosynthesis. Rapid phosphorylation of 60K and 95K proteins has been reported after treatment of chromaffin cells with barium in the absence of calcium (12). The data presented here therefore suggest that phosphorylation of these proteins could be involved in secretion but not biosynthesis of met-enkephalin and VIP. It may be useful to investigate other calcium-dependent processes in the neurendocrine cell with respect to selective stimulation by divalent cations.
10.
11.
12.
13.
14.
Acknowledgements J.W. was a recipient of a National Research Service Award postdoctoral fellowship from the National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Award F32 NS07603. We wish to thank Chang-Mei Hsu for performing the met-enkephalin and VIP radioimmunoassays.
15.
16. 17.
References 1. Viveros, O.H., Diliberto Jr., E.J., Hazem, E. and Chang,
2.
3.
4
5
6
7
8. 9.
K.-J. (1979) Opiate-like materials in the adrenal medulla; evidence for storage and secretion with catecholamines. Mol. Pharmacol. 16: 1101-1108. Livett, B.G., Dean, D.M., Whelan, L.G., Udenfriend, S. and Rossier, J. (1981) Co-release of enkephalin and catecholamines from cultured chromaffin cells. Nature (London) 289: 317-319. Eiden. L.E., Eskay, R.L., Scott, J., Pollard, H. and Hotchkiss, A.J. (1983) Primary cultures of bovine chromaffin cells synthesize and secrete vasoactive intestinal polypeptide (VIP). Life Sci. 33: 687-693. Eiden. K.E., Giraud, P., Dave, J.R., Hotchkiss, A.J., and Affolter. H.-U. (1984) Nicotinic receptor stimulation activates enkephalin release and biosynthesis in adrenal chromaflin cells. Nature (London) 312: 661-663. Siegel. R.E., Eiden, L.E. and Affolter, H.-U. (1985) Elevated potassium stimulates enkephalin biosynthesis in bovine chromaffin cells. Neuropeptides 6543-552. Waschek, J.A., Pruss, R.M., Siegel, R.E., Bade, M.-F., Aunis, D. and Eiden, L.E. (1987) Regulation of enkephalin, VIP and chromogrann in biosynthesis in activity secreting chromaffin cells: multiple strategies for multiple peptides. Ann. NY Acad. Sci. 493: 308-323. Affolter, H.-U., Giraud, P., Hotchkiss, A.J. and Eiden, L.E. (1984) Stimulis-secretion-synthesis coupling: a model for cholinergic regulation of enkephalin secretion and gene transcription in adrenomedullary chromaffin cells. In: Fraioli. F., Isadori, A. and Mazzetti, M. (eds) Opitate Peptides in the Periphery, pp 23-30, Elsevier, Amsterdam. Douglas, W.W. and Rubin, R.P. (1964) Stimulant action of barium on the adrenal medulla. Nature (London) 203: 305307. Grill, V. and Efendic, S. (1984) Stimulation by calcium and
18.
45
barium of somatostatin release. Evidence for lower sensitivity of D- vis-a-vis B- and A-cells. Acta Physiol. Stand 122: 401-407. Douglas, W. W., Taraskevich, P.S., and Tomiko, S.A. (1983) Secretagogue effect of barium an output of melanocyte-stimulating hormone from pars intermedia of the mouse pituitary. J. Physiol. 338: 243-257. Kilpatrick, D.L., Lewis, R.V., Stein, S. and Udenfriend, S. (1980) Release of enkephalin and enkephalin-containing polypeptides from perfused beef adrenal glands. Proc. Natl. Acad. Sci. USA 77: 7473-7475. Amy, C.M.. and Kirschner, N. (1981) Phosphorylation of adrenal medulla cell proteins in conjunction with stimulation of catecholamine secretion. J. Neurochem. 36: 847-854. Holz, R.W., Senter, R.A. and Frye, R.A. (1982) Relationship between Ca’+ uptake and catecholamine secretion in primary dissociated cultures of adrenal medulla. J. Neurothem. 39: 635-646. Wilson, S.P., Chang, K.-J. and Viveros, O.H. (1982) Proportional secretion of opioid peptides and catecholamines from adrenal chromaffin cells in culture. J. Neurosci. 2: 1150-1156. Artalejo, C.R., Garcia, A.G. and Aunis, D. (1987) Chromaffin cell calcium channel kinetics measured isotopitally through fast calcium, strontium, and barium fluxes. J. Biol. Chem. 262: 915-926. Hille. H. (1984) Ionic Channels in Excitable Membranes. Sinaur, Sunderland, Mass. Eiden, L.E., Nilaver, G. and Palkovits, M.P. (1982) Distribution of vasoactive intestinal polypeptide (VIP) in rat brain stem nuclei. Brain Res. 231: 472-477. Eiden, L.E., Giraud, P., Affolter, H.-U.. Herbert, E. and Hotchkiss. A.J. (1984) Alternative modes of enkephalin biosynthesis regulation by reserpine and cyclic AMP in cultured chromaffin cells. Proc. Natl. Acad. Sci. USA 81: 3949-3953.
19. Gubler, U., Seeburg, P., Hoffman, B.J., Gage, L.P. and Udenfriend, S. (1982) Molecular cloning establishes proenkephalin as precursor of enkephaline-containing peptides. Nature (London) 295: 206-208. 20. Kley, N.. Loeffler, J. Ph., Pittius, C.W. and Hollt. V. (1986) Proenkephalin A gene expression in bovine chromaffin cells is regulated by changes in electrical activity. EMBO 5: 967-970. 21 Wilson, S.P. and Kirschner, N. (1983) Calcium-evoked secretion from digitonin-permeabilized adrenal medullary chromafhn cells. J. Biol. Chem. 258: 4994-5000. 22 Pruss. R.M.. Moskal, J.R., Eiden. L.E. and Beinfeld, M.C. (1985) Specific regulation of vasoactive intestinal polypeptide biosynthesis by phorbol ester in bovine chromaffin cells. Endocrinology 117: 1020-102 23 Eiden, L.E. and Hotchkiss, A.J. (1983) Cyclic adenosine monophosphate regulates vasoactive intestinal polypeptide and enkephalin biosynthesis in cultured bovine chromaffin cells. Neuropeptides 4: 1-9. 24. Teo, T.S. and Wang, J.H. (1973) Mechanismsofactivation of a cyclic adenosine 3’:5’-monophosphate phosphodiesterase from bovine heart bv calcium ions. .I. Biol. Chem. 248: 5950-5955. 25. Habermann, E.. Crowell. K. and Janicki, P. (1983) Lead and other metals can substitute for calcium in calmodulin. Arch Toxicol. 54: 61-70. 26. Mills. J.S.. and Johnson, J.D. (1985) Metal Ions as allosteric regulators of calmodulin. J. Biol. Chem. 260: 15105.2222.
Calcium Requirements for Barium Stimulation of Enkephalin and Vasoactive Intestinal Peptide Biosynthesis in Adrenomedullary Chromaffin CellIs JAMES A. WASCHEK
and LEE E. ElDEN
Unit on Molecular and Cellular Neurobiology, Health, National Institutes of Health; Building J. W.1
Laboratory of Cell Biology, National institute of Mental 36, Room 3A-17; Bethesda, MD 20892 (reprint requests to
Abstract-The divalent cation barium was used to study the role of calcium in coupling neuropeptide secretion and biosynthesis following secretagogue stimulation of bovine chromaffin cells. Barium chloride (0.1-2.5 mM) stimulated in a dose-dependent manner the secretion of met-enkephalin (up to 20% of intracellular peptide content) and increased the total amount (cell plus medium content) of met-enkephalin and vasoactive intestinal polypeptide (VIP) 2- to 3-fold after 72 hours. A greater than six-fold increase in proenkephalin mRNA (mRNAenk) was observed by 24 hours following barium stimulation. The voltagesensitive calcium channel blocker D600 inhibited the barium-stimulated secretion of enkephalin and blocked the stimulation of VIP biosynthesis and mRNAenk. Reducing calcium in the medium resulted in an enhancement of barium-stimulated release of both peptides, but blocked the induction of their biosynthesis. The data indicate that calcium targets involved in secretion can be activated by barium or calcium while calcium targets involved in biosynthesis specifically require calcium. It is therefore proposed that pathways leading to peptide secretion and biosynthesis in the adrenal diverge just after secretagogue-stimulated calcium influx.
Introduction
intestinal polypeptide (VIP), by either nicotinic stimulation or depolarization, is accompanied by an increase in peptide biosynthesis, which occurs at a pretranslational locus (4-6). The secretion and enhanced biosynthesis of met-enkephalin and VIP can be inhibited by reducing extracellular calcium or by treatment with agents that block influx of calcium into the cell (3,5-7). The term stimulussecretion-synthesis coupling has been applied to the coordinate calcium-dependent stimulation of both neuropeptide secretion and biosynthesis by
Stimulation of chromaffin cells either with the secretogogue acetylcholine or by depolarization with elevated potassium causes catecholamines and secretory peptides to be released by exocytosis from the vesicles in which they are stored into the extracellular space (l-3). Release of at least two of these peptides, met-enkephalin and vasoactive Date received 16 September 1987 Date accepted 21 September 1987
39
40 chromaffin cell-specific secretagogues (4,7). In the present study, the sites of calcium action within the chromaffin cell are examined using the divalent cation barium, which has been previously used to study secretion in the adrenal medulla and other neuroendocrine tissues (8-14). Barium apparently depolarizes the chromaffin cell (15)) presumably because of its ability to penetrate voltagedependent calcium channels and its ability to block calcium-dependent outwardly-directed potassium current (16). The results presented here indicate that calcium targets for peptide secretion vs. biosynthesis are markedly different in their sensitivity to barium. Materials and Methods Cell Culture. Chromaffin cells were obtained from fresh bovine adrenal medulla and were cultured in a humidified 95% sir/5% carbon dioxide atmosphere as described (3) in non-collagen coated Coastar 24-well dishes (16-mm diameter) at a density of 750000 to 1000000 cells per well in the Basal Medium of Eagle with Earle’s salts and HEPES, 25 mM, supplemented with 5% fetal bovine serum (Hyclone), penicillin 100 units/ml, streptomycin 100 @ml, and cytosine arabinoside 1OpM. After 2 to 3 days in culture, 30 to 60 minutes before adding drugs, medium was removed and replaced with fresh medium diluted 1:l with a solution of chloride salts of sodium, potassium, calcium, and magnesium, at concentrations of 154, 5.4, 1.8, and 0.8 mM, respectively. Replacement of sulfate, carbonate and phosphate salts with the chloride salts in this manner prevented precipitation of barium salts, which occurred in the standard culture medium at barium concentrations greater than 2 mM after 4 to 5 days. The preincubation medium contained D600 (Knoll) at doses indicated or reduced calcium in experiments where barium was to be given under these conditions. In one set of experiments, where bariuminduced met-enkephalin release was measured over several calcium concentrations, a special medium (SRM (3)) which contained no fetal bovine serum (and therefore no added calcium from either supplemental salts or serum) was used for preincubation and measurement of peptide release. Immediately before experiments, preincubation medium was removed and replaced with identical medium containing various concentrations of barium. Release experiments were carried out over a 30-minute time interval unless otherwise specified. Biosynthesis experiments
NEUKOPEPTIDES
were carried out for 24 hours (mRNA levels) and 72 hours (peptide levels). Peptide radioimmunoassay.
Met-enkephalin and VIP immunoreactivity in medium and cell extracts were measured as described (3) using metenkephalin antiserum (RB-4) kindly provided by Dr. Steve Sabol (NHLBI, National Institutes of Health) and VIP antiserum N12 kindly provided by Dr.. Gajanan Nilaver, Columbia University College of Physicians and Surgeons (17). mRNAenk quantitation. RNA was extracted from cells, denatured, and electrophoresed on agarose gels as described (18). Total RNA extracted was estimated by densitometric scanning of the 28s band appearing on photographic negatives of UV illuminated gels stained with ethidium bromide. The RNA was then transblotted from the gel to Gene Screen (NEN) and hybridized with a nicktranslated 400 base-pair Pst I fragment of the cDNA encoding bovine preproenkephalin A (19). mRNAenk was quantitifed by densitometric scanning of film exposed to the hybridized blots. In some cases, after total RNA measurement, mRNAenk was quantified by direct slot blot RNA application because hybridization to nonmRNAenk species was negligible.
Results Release of met-enkephalin by barium chloride was dose dependent and was maximal (22% of cell content) at 4 mM (Fig. 1). To determine whether or not barium acted directly at calcium sites or indirectly by facilitating the action of calcium, cells were treated with barium, 1 mM and 2.5 mM, in the presence of normal (1.8 mM) and reduced (0.1 mM) concentrations of extracellular calcium and in the presence of D600, which blocks voltagedependent calcium channels. Release at both barium concentrations was enhanced in medium with reduced calcium, and was inhibited by D600 (Fig. 2). Release of met-enkephalin from cells treated with 2 mM barium over a range of calcium concentrations was maximal in the absence of added calcium and was reduced several-fold at a calcium concentration of 10 mM (Fig. 3). The release of VIP elicited by 2 mM barium was likewise inversely related to the extracellular calcium concentration, and was maximal in the total absence of added extracellular calcium (Table 1). Thus, barium probably enters the cell through the D600-sensitive calcium channel and
CALCIUM
REQUIREMENTS
FOR BARIUM
STIMULATION
-11
OF ENKEPHALIN
Table 1 Inhibation of Barium-Stimulated Release of Vasoactive Intestinal Polypeptide (VIP) in Bovine Chromaffin Cells by Extracellular Calcium Concentration of CaCl, added
ControI BaCI,, 2mM
OmM
2.2 mM
20.5 f 2.7 99.7 * 6.6
23.5 + 5.6 59.3 + 2.5
10mM 16.3 i 10.1 39.2 + 3.2
Cells were preincubated for 1 hr at 37” in serum-free medium containing the indicated concentration of added calcium chloride. Concentration of VIP in the medium was measured 1 hr after addition of barium (values are pg/well. mean f SEM. n= 3).
253
20,
Fig 2 Barium-stimulated release of enkephalin by bovine adrenal chromaffin cells over 30 minutes (% of cell contents ? SEM. n=3) in normal (N) medium (1.8 mM Ca”), in low calcium (LC) medium (100 uM). and in normal medium containing the calcium channel blocker, D600, 10 PM. Cells were preincubated for 1 hr in the indicated medium before addition of 1 mM barium. Mean total met-enkephaiin content/ well (cells plus medium) was 2.7 to 2.9 ng. T = p< .05 compared with respective value in control group. 1 = p<.O5 compared with barium control (Student’s t-test).
3 E ae 15r
0
70 -
1.0
2.0
3.0
4.0 60-
Barium Cont., mM Fig 1 Release affin cells over of barium (% met-enkephalin “g.
of met-enkephalin by bovine adrenal chrom30 minutes after stimulation with several doses of cell contents, 2 SEM, n=3). Mean total content/well (cells plus medium) was 3.7 to 4.1
causes met-enkephalin
and VIP release by acting directly at intracellular calcium sites. Met-enkephalin and VIP levels (cells plus medium) increased with time following treatment with 2.5 mM barium; increases in both peptides were fully apparent after 72 hours (Fig. 4). Maximal elevation of both peptides at 72 hours was observed with 1 mM barium (Fig. 5). Induction of met-enkephalin peptide by barium occurred in association with increased levels of mRNAenk measured at 24 hours (Fig. 6), in agreement with a recent report (20). Thus, barium appears to stimulate met-enkephalin biosynthesis at a pretranslational locus, as do nicotine and depolarizing concentrations of potassium ion (4,5). Barium induction of met-enkephalin and VIP peptide and mRNAenk (Fig. 7) was inhibited by reducing extracellular calcium. This was in con-
504O3020 10 0.
’
No calcium
// N 5.0
-log
I
I
4.0
3.0
-
9 2.0
[Ca2+I, M added
Fig 3 Release of met-enkephalin by bovine adrenal chromaftin cells over 60 minutes 1% of cell contents + SEM, n=3) at several concentrations of extracellular calcium when treated with (0) or without (0) barium, 2 mM. Mean total metenkephalin content/well (cells plus medium) was 2.0 to 2.7 ng.
trast to the enhancement of barium-stimulated secretion caused by this treatment, and suggest that calcium targets for these processes are different. Induction of VIP, but not metenkephalin peptide or mRNAenk by barium was blocked by 10 PM D600 (Table 2). D600 did
NEUROPEPTIDES
12r
0.0
C
Ba
C
15 hr
Ba
40 hr
C
Ba
72 hr
Fig 4 Time-course in increase in cell and medium VIP (upper frame) and met-enkephalin (lower frame) after treatment of bovine chromaffin cells with barium, 2.5 mM over 72 hours. Peptide contents measured in the cell and medium are indicated by shaded and open bars, respectively. SEM values are for cell peptide and total peptide contents (n=3).
Table 2
Effect
Proenkephalin
Control(d) Barium 1 mM Barium 1 mM +D600 10 uM Barium 1 mM +D600 30 PM
0.1
0.2
0.5
Barium concentration,
1.0
2.0
mM
Fig 5 Total (cell and medium) VIP (upper frame) and met-enkephalin (lower frame) 72 hours after treatment of bovine chromaftin cells with several doses of barium. Peptide contents measured in the cell and medium are indicated by shaded and open bars, respectively. SEM values (n=3) are for cell peptide and total peptide contents. 1 = p<.O5 compared with untreated control value (Student’s t-test).
of D600 on 1 mM Barium Induction of Met-Enkephalin and VIP Levels after 72 Hours and mRNA after 24 Hours Treatment of Bovine Chromaffin Cells(a) VIP(b)
met-enkephalin(b)
51.8 f 1.5 121.4 + 2.7(Y) 40.3 zb 3.3(Y,l)
3618 f 357 10439 f 387(Y) 10199 + 361(Y)
Proenkephalin
1.0 + 0.1 4.2 + 0.5(Y) 4.3 f 0.3(V)
mRNA(c)
n.d.
nd.
2.0 f 0.3(Y.1)
(a) Values are mean + SEM, n=3 for all treatments except barium 1 mM alone (n=9) (b) Total contents in cells and medium, pg/well. (c) Standarized to total RNA extracted and normalized to control value. Data are pooled from 3 separate experiments, n=3 to 9. (d) Control values of D600 were not significantly lower than untreated controlvalues. (Y) pc.05 compared with control group. (7) p-z.05 compared with barium alone group. (n.d.) not determined
CALCIUM
REQUIREMENTS
FOR BARIUM
STIMULATION
43
OF ENKEPHALIN
Barium, 1 mM
Y
120 80 - Barium, 0 mM 40.
01
’
0.0
1
1
I
1.0
2.0
3.0
1
I
4.0
O-
N LC
N LC
N LC
N LC
Barium Concentration, mM Fig 6 Concentration of mRNA’“’ in bovine chromaffin cells (relative to control) measured 24 hours after treatment with several doses of barium. Individual values (fSEM, n=3) are corrected for total RNA extracted, as described in methods.
inhibit the barium-stimulated increase in mRNAenk at 30 PM, a dose higher than that needed to inhibit barium stimulated metenkephalin secretion (Fig. 2), or to inhibit induction of met-enkephalin peptide and mRNA by nicotine or depolarizing concentrations of potassium (4,6,7). Discussion The data presented here demonstrate that barium stimulates both release and biosynthesis of neuropeptides in chromaffin cells, consistent with previous studies on secretion in bovine chromaffin cells (8,11-14), and with a recent report showing enkephalin mRNA induction by barium (20). The data here further show that biosynthesis of VIP can also be stimulated by barium, and that barium cannot fully substitute for calcium in stimulation of biosynthesis of either peptide. Thus, barium stimulates biosynthesis of these peptides indirectly, presumably by depolarizing the cell and allowing calcium to enter the cell to act at the appropriate intracellular sites. Secretion, on the other hand, was elicited in the complete absence of added extracellular calcium, indicating that barium probably acted directly at calcium sites to stimulate secretion. It could be argued that barium transiently increased intracellular calcium by displacing it from intracellular stores to cause secretion. However, a corresponding effect on biosynthesis was not observed. Additional support for a direct agonist action of barium in secretion is that barium is able to produce caIcium-independent secretion from permeabilized chromaffin cells (21). In any
12 8 4 0
6-
Barium 1.0 mM Y
Barium, 0 mM
N LC
N LC
Fig 7 Effect of medium containing reduced calcium on the barium-stimulated biosynthesis of VIP and met-enkephalin in bovine chromaffin cells. Total peptide levels (cell and medium) of VIP (upper) and met-enkephalin (middle) after 72 hours treatment and proenkephalin mRNA levels (lower) after 24 hours treatment with 1 mM barium in normal (N) medium (1.8 mM Ca2+), or in low calcium (LC) medium (100 uM CaZ+). Peptide contents measured in the cell and medium in upper two panels are indicated by darkened and open bars, respectively, with SEM values referring to cell and total peptide contents. Proenkephalin mRNA values (lower panel), corrected for total RNA extracted as described in the methods, are normalized to control values. N = 3 for all peptides experiments; n = 6 to 9 for RNA experiments (pooled data from 2 experiments). f = p<.O5 compared with respective value in control group. 7 = p<.O5 compared with barium control value (Student’s t-test).
44
NEUROPEPTIDES
event, it is shown here that barium distinguishes calcium-dependent processes involved in secretion versus biosynthesis of met-enkephalin in chromaffin cells. Biosynthesis and release of another peptide, VIP, was also stimulated by barium. Induction of VIP biosynthesis by barium was more sensitive to D600 than was induction of enkephalin. VIP appears to be inducible in a small subset of cells distinct from those that express met-enkephalin (22). This differential regulation thus appears to be exhibited in two different cell types, and may reflect differences at the cell membrane or at intracellular sites. Induction of VIP by nicotine does appear to be at the level of mRNA induction because increased peptide levels are blocked by cycloheximide (6). A direct comparison of effects on met-enkephalin and VIP mRNA could not be made because a polynucleotide probe to measure bovine VIP mRNA is not currently available. Cyclic AMP as well as calcium may be involved in the biosynthesis of met-enkephalin and VIP in
chromaffin cells (23). Since nicotine stimulation of met-enkephalin biosynthesis is calcium dependent, and nicotine causes an increase in intracellular CAMP (4,7,23), it is possible that calcium and CAMP may act cooperatively to stimulate biosynthesis or that calcium acts via an increase in CAMP. An additional possibility is that CAMP is important in regulating enkephahn and VIP in neuroendocrine cells during expression development, but is not required for stimulussecretion-synthesis coupling in the mature chromaffin cell. The data presented here and in the literature are consistent with the model for the role of calcium in secretagogue-stimulated peptide release and the compensatory increase in peptide biosynthesis Calcium voltage-sensitive calcium channels. enters the cell and activates factors associated with secretion and other distinct factors associated with regulation of peptide biosynthesis. Since barium appears to bind calmodulin poorly and activates some calmodulin-mediated processes weakly if at
Paptide
biosynthesis
Ca*+ 0 Ba** n
Peptide
secretion
Fig 8 Model depicting the interaction of calcium and barium with regulatory factors involved in peptide secretion and the compensatory increase in biosynthesis of secreted peptides in bovine chromaffin cells. Following stimulation with acetylcholine or by depolarization, calcium enters the cell through voltage-sensitive calcium channels and stimulates peptide secretion and biosynthesis by interacting at the distinct calcium binding sites that govern these processes. Barium stimulates peptide release by directly binding and activating calcium targets associated with secretion, but stimulates biosynthesis only by facilitating the action of calcium. Barium may facilitate the action of calcium by depolarizing the cell and allowing calcium entry into the cell through voltage-dependent calcium channels.
CALCIUM
REQUIREMENTS
FOR BARIUM
STIMULATION
OF ENKEPHALIN
all (24-26), the data here argue against an important regulatory role for calmodulin in chromaffin cell secretion, but are not inconsistent with a role of calmodulin in peptide biosynthesis. Rapid phosphorylation of 60K and 95K proteins has been reported after treatment of chromaffin cells with barium in the absence of calcium (12). The data presented here therefore suggest that phosphorylation of these proteins could be involved in secretion but not biosynthesis of met-enkephalin and VIP. It may be useful to investigate other calcium-dependent processes in the neurendocrine cell with respect to selective stimulation by divalent cations.
10.
11.
12.
13.
14.
Acknowledgements J.W. was a recipient of a National Research Service Award postdoctoral fellowship from the National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Award F32 NS07603. We wish to thank Chang-Mei Hsu for performing the met-enkephalin and VIP radioimmunoassays.
15.
16. 17.
References 1. Viveros, O.H., Diliberto Jr., E.J., Hazem, E. and Chang,
2.
3.
4
5
6
7
8. 9.
K.-J. (1979) Opiate-like materials in the adrenal medulla; evidence for storage and secretion with catecholamines. Mol. Pharmacol. 16: 1101-1108. Livett, B.G., Dean, D.M., Whelan, L.G., Udenfriend, S. and Rossier, J. (1981) Co-release of enkephalin and catecholamines from cultured chromaffin cells. Nature (London) 289: 317-319. Eiden. L.E., Eskay, R.L., Scott, J., Pollard, H. and Hotchkiss, A.J. (1983) Primary cultures of bovine chromaffin cells synthesize and secrete vasoactive intestinal polypeptide (VIP). Life Sci. 33: 687-693. Eiden. K.E., Giraud, P., Dave, J.R., Hotchkiss, A.J., and Affolter. H.-U. (1984) Nicotinic receptor stimulation activates enkephalin release and biosynthesis in adrenal chromaflin cells. Nature (London) 312: 661-663. Siegel. R.E., Eiden, L.E. and Affolter, H.-U. (1985) Elevated potassium stimulates enkephalin biosynthesis in bovine chromaffin cells. Neuropeptides 6543-552. Waschek, J.A., Pruss, R.M., Siegel, R.E., Bade, M.-F., Aunis, D. and Eiden, L.E. (1987) Regulation of enkephalin, VIP and chromogrann in biosynthesis in activity secreting chromaffin cells: multiple strategies for multiple peptides. Ann. NY Acad. Sci. 493: 308-323. Affolter, H.-U., Giraud, P., Hotchkiss, A.J. and Eiden, L.E. (1984) Stimulis-secretion-synthesis coupling: a model for cholinergic regulation of enkephalin secretion and gene transcription in adrenomedullary chromaffin cells. In: Fraioli. F., Isadori, A. and Mazzetti, M. (eds) Opitate Peptides in the Periphery, pp 23-30, Elsevier, Amsterdam. Douglas, W.W. and Rubin, R.P. (1964) Stimulant action of barium on the adrenal medulla. Nature (London) 203: 305307. Grill, V. and Efendic, S. (1984) Stimulation by calcium and
18.
45
barium of somatostatin release. Evidence for lower sensitivity of D- vis-a-vis B- and A-cells. Acta Physiol. Stand 122: 401-407. Douglas, W. W., Taraskevich, P.S., and Tomiko, S.A. (1983) Secretagogue effect of barium an output of melanocyte-stimulating hormone from pars intermedia of the mouse pituitary. J. Physiol. 338: 243-257. Kilpatrick, D.L., Lewis, R.V., Stein, S. and Udenfriend, S. (1980) Release of enkephalin and enkephalin-containing polypeptides from perfused beef adrenal glands. Proc. Natl. Acad. Sci. USA 77: 7473-7475. Amy, C.M.. and Kirschner, N. (1981) Phosphorylation of adrenal medulla cell proteins in conjunction with stimulation of catecholamine secretion. J. Neurochem. 36: 847-854. Holz, R.W., Senter, R.A. and Frye, R.A. (1982) Relationship between Ca’+ uptake and catecholamine secretion in primary dissociated cultures of adrenal medulla. J. Neurothem. 39: 635-646. Wilson, S.P., Chang, K.-J. and Viveros, O.H. (1982) Proportional secretion of opioid peptides and catecholamines from adrenal chromaffin cells in culture. J. Neurosci. 2: 1150-1156. Artalejo, C.R., Garcia, A.G. and Aunis, D. (1987) Chromaffin cell calcium channel kinetics measured isotopitally through fast calcium, strontium, and barium fluxes. J. Biol. Chem. 262: 915-926. Hille. H. (1984) Ionic Channels in Excitable Membranes. Sinaur, Sunderland, Mass. Eiden, L.E., Nilaver, G. and Palkovits, M.P. (1982) Distribution of vasoactive intestinal polypeptide (VIP) in rat brain stem nuclei. Brain Res. 231: 472-477. Eiden, L.E., Giraud, P., Affolter, H.-U.. Herbert, E. and Hotchkiss. A.J. (1984) Alternative modes of enkephalin biosynthesis regulation by reserpine and cyclic AMP in cultured chromaffin cells. Proc. Natl. Acad. Sci. USA 81: 3949-3953.
19. Gubler, U., Seeburg, P., Hoffman, B.J., Gage, L.P. and Udenfriend, S. (1982) Molecular cloning establishes proenkephalin as precursor of enkephaline-containing peptides. Nature (London) 295: 206-208. 20. Kley, N.. Loeffler, J. Ph., Pittius, C.W. and Hollt. V. (1986) Proenkephalin A gene expression in bovine chromaffin cells is regulated by changes in electrical activity. EMBO 5: 967-970. 21 Wilson, S.P. and Kirschner, N. (1983) Calcium-evoked secretion from digitonin-permeabilized adrenal medullary chromafhn cells. J. Biol. Chem. 258: 4994-5000. 22 Pruss. R.M.. Moskal, J.R., Eiden. L.E. and Beinfeld, M.C. (1985) Specific regulation of vasoactive intestinal polypeptide biosynthesis by phorbol ester in bovine chromaffin cells. Endocrinology 117: 1020-102 23 Eiden, L.E. and Hotchkiss, A.J. (1983) Cyclic adenosine monophosphate regulates vasoactive intestinal polypeptide and enkephalin biosynthesis in cultured bovine chromaffin cells. Neuropeptides 4: 1-9. 24. Teo, T.S. and Wang, J.H. (1973) Mechanismsofactivation of a cyclic adenosine 3’:5’-monophosphate phosphodiesterase from bovine heart bv calcium ions. .I. Biol. Chem. 248: 5950-5955. 25. Habermann, E.. Crowell. K. and Janicki, P. (1983) Lead and other metals can substitute for calcium in calmodulin. Arch Toxicol. 54: 61-70. 26. Mills. J.S.. and Johnson, J.D. (1985) Metal Ions as allosteric regulators of calmodulin. J. Biol. Chem. 260: 15105.2222.