Neurochem. Int. Vol. 20, No. 4, pp. 511-519, 1992 Printed in Great Britain. All rights reserved
019%0186/9255.00+0.00 Copyright © 1992 Pergamon Press Ltd
INORGANIC THIOPHOSPHATE EFFECTS ON CHROMAFFIN CELL STRUCTURE AND FUNCTION JACK C. BROOKS,I* MELINDA H. BROOKSI and STEPHEN W. CARMICHAEL2 LMarquette University School of Dentistry, Department of Basic Sciences, 604 North 16th Street, Milwaukee, WI 53233, U.S.A. 2Department of Anatomy, Mayo Clinic, Medical Science Bldg. 3, Rochester, MN 55905, U.S.A. (Received 20 May 1991 ; accepted 9 October 1991)
Abstract--The effect was determined of replacing medium inorganic phosphate with thiophosphate on the structure and function of cultured bovine chromaffin cells. Cell cultures were incubated in normal medium containing fetal bovine serum, phosphate free medium or similar medium supplemented with inorganic phosphate or thiophosphate. In contrast to the other media, cells cultured with thiophosphate medium for 3~, days showed seriously compromised structure and functions. The cells lost 75% of their catecholamine content and their ability to secrete remaining catecholamines in response to nicotine stimulation. Radiolabelled thiophosphate was rapidly taken up by the cells and, in long-term experiments, was incorporated largely into a 97 121 kDa protein band on SDS-PAGE. Additional minor bands were found to a lesser, variable extent. Transmission electron micrograpbs of cells treated with thiophosphate showed extensive depletion of chromaffin vesicles and disruption of mitochondria, suggesting that the functional damage noted with these cells could be associated with damage to mitochondria. Analysis of general cell metabolic activity by conversion of the dye (3-[3,4-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide) to its formazan derivative indicated increased metabolic activity at early stages of exposure to thiophosphate followed by a decline with continued exposure, supporting the argument for an overall depression of cell metabolism. Uptake of the dye neutral red, which is avidly accumulated by chromaffin cells, was also reduced for cells exposed to thiophosphate. The data suggest that thiophosphate enters chromaffin cells and disrupts energy dependent cell functions, including catecholamine storage and secretion.
N u m e r o u s studies have suggested a role for protein phosphorylation in the process of catecholamine secretion from chromaffin cells (Amy and Kirshner, 1981 ; Cote et al., 1986 ; Lee and Holz, 1986 ; Haycock et al., 1988a,b; Brooks and Brooks, 1987a,b; Burgoyne, 1984). Many of these experiments share the c o m m o n protocol of incubating the cultured cells with 32Pi to permit them to incorporate it into ATP. The [7-32p]ATP is then available as substrate for protein phosphorylation reactions and these reactions are correlated with elicited cellular responses. Numerous proteins are phosphorylated in chromaffin cells, in association with secretion, in either intact or permeabilized cells. A recent study (Gutierrez et al., 1988) used this protocol with two-dimensional electro-
phoresis to map over 500 phosphoproteins in chromaffin cells. The A T P analog ATPTS (adenosine-5'-O-(3-thiotriphosphate)) also serves as a substrate for (thio)phosphorylation reactions. However, protein thiophosphorylation reactions are generally considered irreversible because cellular phosphatases remove the thiophosphoryl groups very slowly or not at all (Cassidy et al., 1979; Sherry et al., 1978; Coyne et al., 1987). Our strategy has been to treat permeabilized chromaffin cells with ATPTS to lock phosphorylation reactions in the thiophosphorylated state. This then permits us to evaluate the relationship between such reactions and the secretory process. Secretion by permeabilized cells is inhibited by exposure to ATPTS and is correlated with the thiophosphorylation of several proteins (Brooks and Brooks, 1985, 1987a,b). The data have led us to the hypothesis that a phos*Author to whom correspondence should be addressed. phorylation reaction may be important in the control Abbreviations: MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diof secretion, although a causal relationship has not phenyltetrazolium bromide) ; MTT formazan, (l-[4,5dimethylthiazole-2-yl]-3,5-diphenylformazan), TP~, thio- been established. In the experiments reported here, we have treated phosphate ; ATP?S, adenosine-5'-O- (3-thiotriphosphate). intact chromaffin cells with inorganic thiophosphate 511
JACK C. BROOKS el al.
512
using a p r o t o c o l a n a l o g o u s to t h a t used in e x p e r i m e n t s with i n o r g a n i c p h o s p h a t e . T h e objective was to force the cells to synthesize ATPTS so that p h o s p h o r y l a t i o n reactions c o u l d be s t u d i e d in intact cells. W e h y p o t h e s i z e d t h a t the t h i o p h o s p h a t e w o u l d be t a k e n up by the cells a n d synthesized into ATPTS, w h i c h w o u l d t h e n be used for p r o t e i n p h o s p h o r y l a t i o n reactions. T h e e x p e c t a t i o n was t h a t we could devise specific t r e a t m e n t r e g i m e n s w h i c h w o u l d allow us to identify the role o f various p h o s p h o r y l a t i o n reactions in cell functions. In particular, p r o t e i n s essential to secretion m i g h t be p e r m a n e n t l y t h i o p h o s p h o r y l a t e d a n d affect the cell secretory response.
EXPERIMENTAL PROCEDURES
Materials All reagents were analytical grade or the best available grade. Dulbecco's modified Eagle Medium, HAM F-12 Nutrient Mixture and antibiotics were purchased from GIBCO (Grand Island, NY). Dulbecco's Modified Eagle's Medium (High glucose, phosphate-free, HEPES-buffered) was obtained from Irvine Scientific (Santa Ana, CA) as a powder, prepared and filter sterilized before use. This medium was designated as P~-free medium; it was supplemented with 3 mM Na3PO4 (3 mM Pi-medium) or 3 mM Na3PO3S" 12H20 (3 mM TPi-medium) for cell culture. The initial pH of all media was adjusted to 7.4. Carrier free sodium thiophosphate, specific activity 48.1 TBq/mmol, was purchased from New England Nuclear lnc., Boston, MA. Nicotine, MTT, MTT formazan, neutral red (purified), Hoechst 33258 (bisbenzamide) and calf thymus DNA were obtained from Sigma Chemical Co., St Louis, MO. Tris (gold label) was obtained from Aldrich Chem. Co., Milwaukee, W1. An electrophoresis calibration kit for low molecular weight proteins was obtained from Pharmacia (Piscataway, N J). Other electrophoresis chemicals were obtained from Polysciences Inc., Warren, PA. General experimental protocol Bovine chromaffin cells were prepared and maintained in primary culture as described previously (Brooks and Treml, 1983). Isolated cells were suspended at 106 cells/ml and plated at 2 x 10 s cells/well in 96 well culture plates (Falcon, 3070) or, for experiments involving protein thiophosphorylation, at 106 cells/well in 24 well plates (Corning, Cell Wells 25820). The medium was changed after 4 days and the cells were used between 4 and 8 days of culture. Cytosine arabinoside was used at 10/tM concentration in the culture medium to inhibit fibroblast growth. The cultures contained about 85% chromaffin cells. Celt monolayers used for experiments were washed 3 x 5 min. with 0.2 ml/well of complete Lockes solution (Brooks and Treml, 1983), 0.9% saline or Pi-free medium, as required by the experiment. The cells were then exposed to 0.2 ml/well of treatment solutions. In some experiments Lockes solution was prepared without phosphate and supplemented with either 3 mM P, or TP~. This was used to determine the effect of TP~ presence in the medium during stimulated secretion. Secretion was elicited with 20 itM nicotine. MTT was solu-
bilized in Tris buffered saline (50 mM Tris CI. 0.145 M NaCI, pH 7.4).
Analytical methods Total cellular catecholamine content and catecholamine released to the medium during assay of secretion was determined fluorometrically as described previously (Brooks and Treml, 1983). The catecholamine content of individual samples was determined from a standard curve relating relative fluorescence to known concentrations of a 1:I mixture of norepinephrine and epinephrine. For determination of total cellular catecholamine content, cell monolayers were dissolved in 0.2 ml/well of 0.01 M HC1. Data are expressed as ng catecholamine/2 x 105 cells as indicated in the tables. Since high concentrations of thiophosphate interfered slightly with the fluorometric assay, a standard curve was constructed in the presence of 3 mM TP~ for measurements of secretion made in media containing thiophosphate. Total DNA conlent of monolayers was used to determine if experimental conditions resulted in the loss of viable cells. The method used was that of Cesarone et al. (1979), using calf thymus DNA as a standard. DNA standards and monolayers were dissolved in the specified citrate buffer containing 1% SDS (100 #1/well, plus one 100 #1 wash) so that the final concentration of SDS in the assay mixture did not exceed 0.05 %. A fluorometric assay based upon the intrinsic fluorescence of neutral red was designed for the determination of neutral red accumulation by cells. The excitation and emission wavelengths used were 510 and 600 nm, respectively. A standard curve was constructed using relative fluorescence against known concentrations of neutral red in a solution containing t% SDS and 0.01 M HCI; the curve was linear over the range of 0.5 25 ng neutral red/ml. For assay of neutral red accumulation, monolayers of cells incubated with various trcatmem media were washed quickly three times with 0.2 ml/well of 0.9% saline at 3 7 C and incubated for 10 rain with a 0.6 mg/ml solution of neutral red in saline. The cells were then washed four times with 0.2 ml/well of saline and dissolved in 1% SDS in 0.0l M HC1 for determination of neutral red accumulation. The conversion of MTT to MTT formazan was used to determine the metabolic effect and possible cytotoxicity of the different media. The metabolic activity of cells cultured for periods of l 3 days with various media was assayed as described by Magilavy and Rothstein (1988). At the end of the culture period, 100 #1 of medium was removed from each well, 20 #1 of MTT solution (5 mg MTT/ml TBS) was added and the plates returned to the incubator for 1 h. The cells were dissolved at the end of this period by addition of 100 #1/well of 10% SDS in 0.01 M HCI. The plates were returned to the incubator overnight to dissolve the blue MTT formazan reaction product. The formazan was quantitated spectrophotometrically at 570 nm (690 nm reference) on a Minireader-ll microelisa reader (Dynatech Laboratories Inc., Alexandria, VA) against a standard curve relating A , ~ to concentration of MTT formazan. Medium (without cells) was carried through all culture periods to serve as blanks for the assays. Statistical analysis was performed by one-way ANOVA on a VAX system using SPSSX (SPSS Inc.. Chicago, IL) at a significance level of P < 0.05. Determination q/thiophosphate uptake and protein thiophospharvlation For experiments involving the determination of [~S]TP~ uptake by cells, treatment solutions were removed and the
Thiophosphate in chromaffin cells monolayers washed quickly four times with 0.2 ml/well of ice-cold treatment solutions similar to those used in the experiment but free of [35S]TP~. The cells were dissolved in 50/A/well of SDS sample buffer (without mercaptoethanol) used for SDS-PAGE, combined with two similar washes and counted in 7 ml of Aquasol-2 (New England Nuclear Inc., Boston, MA) as previously described (Brooks and Brooks, 1987a). Thiophosphorylation of cellular proteins was determined by [3SS]TPi incorporation into proteins from P~-free medium or media containing P~ or TPI and 20 #Ci/ml of [35S]TPi. After removal of the medium, the monolayers were dissolved directly in sample buffer and subjected to SDSPAGE in 10% polyacrylamide gels (Brooks and Brooks, 1985). Two-dimensional electrophoresis was carried out as described by Hochstrasser et al. (1988). The second dimension SDS-PAGE gels were stained with colloidal Coomassie brilliant blue G-250 (Neuhoff et al., 1988). Proteins thiophosphorylated with [35S]TPi were detected by fluorography for 2-8 day exposures at - 7 0 ° C on Kodak XOmat AR film after treatment of the Coomassie blue stained gel with sodium salicylate (Chamberlain, 1979). Gels were run in a Bio-Rad Mini-Protean system using gel and buffer formulations (SDS-PAGE) provided with the apparatus. The molecular weights of the separated proteins were determined from a curve relating the log molecular weight to migration distance for proteins of known molecular weight. Electron microscopy Cells cultured with the various media were subjected to transmission electron microscopy as previously described (Brooks and Carmichael, 1987). The ceils were cultured for 1 3 days on Lux Thermanox membranes (Miles Laboratories, Naperville, IL) in Micro slide chambers (Bellco Glass, Vineland, N J). The medium was removed and the cells (on the Lux membrane) placed in a freshly prepared solution of 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 at room temperature and transferred to a cold room at 4°C for 1 h. The cells were then washed briefly three times with icecold 0.3 M sucrose buffered with 0.I M cacodylate buffer, pH 7.2. The cells were treated with 1% osmium tetroxide for 1 h, dehydrated, and embedded. Sections were cut on a plane perpendicular to the Lux membrane surface, mounted on copper grids and stained with uranyl acetate and lead citrate. The specimens were examined with a Philips CM 12 electron microscope at 60 kV accelerating voltage.
RESULTS T P i d a m a g e to c a t e c h o l a m i n e content a n d secretion
A n initial m e d i u m concentration o f TP~ was chosen arbitrarily as 3 m M ; this is a b o u t three times the normal medium P~ concentration. The effect o f chronic exposure o f the cells to normal m e d i u m (F12, Dulbecco's Modified Eagle's Medium, 10% fetal bovine s e r u m ; see Brooks and Treml, 1983), P~-free medium and media supplemented with either 3 m M P~ or TP~ is shown in Table 1. N o r m a l medium is that customarily used for primary culture o f chromaffin cells and was included as a reference to which all other media were c o m p a r e d for normal cell function. The P~-free m e d i u m was included as a reference for the
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Table 1. Effectof chronic exposureof chromaffincells to elevatedP~ or TP~on cellcatecholaminecontent and secretion Exposure time
Catecholaminesecretionand cellularcontent (ng/2 x 105 cells) Normal Pl-free 3 mM Pi 3 mM TP~
Day 1"
128.0+6.5 123.3+__7.1 117.5+4.1 55.9 + 2,2:~ 372.2_+12.9 345.8+_8.4 336.4+_7.6 138.3_+8.2 3618_+56 3244+64 3348_+113 1392±50t
Day3
159.9-+4.2 147.9_+5.1 169.7_+3.2 45.7_+2.2:~ 620.0_+21.9 574.8_+6.4 505.0_+13.7 92.3_+9.6 3778_+80 3585_+104 4070+-135 1013_+54"I"
Day 4
255.7+47.3 249.6_+4.2 234.2_+9,0 65.1_+4.5~ 635.8+-17.8 775.1_+20.6 613.1+-15.1 76.0+_6.7 4464_+115 4460_+146 4072_+132 1077_+55"t
Chromaffin cells were cultured in normal medium for 4 days after isolation. The monolayerswere then exposed to 200 #l/well of the treatment solutions and incubated for the periods indicated in the table. For assay, the monolayerswere washed 3 × 5 min with 200 /~l/well of complete Lockes solution at 37°C. Total cellular catecholaminecontent was determined on aliquots of cellsdissolvedin 0.01 M HCI. Secretionwas assayedby exposing cellsto 200/ll/wellof Lockessolution(control)or Lockessolution +20/~M nicotine (stimulated) for 10 min at 37°C. The medium was recoveredand an aliquot taken for determinationof secreted catecholamines. *Under each treatment period, the values listed are unstimulated secretion, stimulated secretion and total cellular catecholamine content. Each table value is the mean+ SEM for 6 (secretion)or 8 (total catecholamine)replicates. ]'Significantly decreased compared to other total catecholamine groups at P < 0.05. :~Significantlydecreasedcompared to other unstimulated groups at P < 0.05. For day 4, the respectivestimulatedgroup is not different from the unstimulatedgroup. effect o f exogenous P~ and TP~ o n cell behavior. Total cellular catecholamine content was reduced by nearly 40% after one day o f exposure to TP~ medium. The absolute magnitude o f catecholamine secretion was similarly reduced, although the ratio o f stimulated to unstimulated release was not different from that o f cells exposed to any o f the other media. After 3 days o f culture the total cellular catecholamine content was reduced to about 25% that o f the other treatment groups and remained at that level through day 4 o f culture. Total secretion was markedly reduced by day 3 o f culture and stimulated secretion was lost by day 4 o f culture even though the cell catecholamine content had stabilized at a b o u t 25% o f normal. The effects o f chronic exposure to elevated TP~ suggested that reduced concentrations might have a similar effect and that perhaps the cells could recover from TP~-induced damage if they were returned to normal medium after a 3 day exposure to TPi medium. Cells were exposed (data not shown) to normal, P~-free medium or medium supplemented with 1-3 m M P~ or TP~ for 3 days. TP~ at 1 m M concentration had no significant effect on either total cellular catecholamine content or secretion over the 3 day exposure period,
514
JACK C. BROOKS el ~ll.
while 2 and 3 mM TP~ significantly reduced total cellular catecholamine content and secretion. Cells exposed to those media for 3 days were returned to normal medium for an additional 3 day period to determine if the TP,-treated cells would recover both their normal catecholamine content and secretory responsiveness. The catecholamine content of all groups remained the same as it was before the cells were returned to normal medium. The cells originally cultured with TP, medium showed no significant improvement in total cellular catecholamine content or stimulated secretion upon return to normal medium. Exposure of cells to 3 mM TP~ in Lockes solution for a 2 h period had no effect on the subsequent secretory responsiveness of the cells in Lockes solution free of TPj. However, stimulated secretion was significantly reduced when the cells were pretreated for 2 h with either 3 mM TPi or P~ in Lockes solution but then stimulated by 20 JiM nicotine in the presence of TP, (not shown). This result suggested that TP~ might have a direct inhibitory effect on secretion and might influence secretion simply by being present during the usual 10 min assay period. The presence of 3 mM TP~ in the assay medium caused a slight reduction of stimulated secretion (Table 2) and a slight increase in unstimulated release when compared to 3 mM P~ medium. The slight inhibitory effect of TPL when present during the assay of secretion suggested that TP~ might enter the cell and affect secretion. If true, this should be reflected in altered uptake of [3SS]TP~ from media. Data describing the uptake of [-~SS]TP, (10 tlCi/ml) from various media is shown in Table 3. Uptake of radiolabel was similar for normal and 3 mM P~ medium for respective unstimulated and stimulated Table 2. Effect of varying phosphale and thiophosphate on catecholamine secretion
Treatmenl P j r c e Lockes 3 m M P,-Lockes 3 m M TPi-Lockes
Catecholamine secretion (ng/2 × 10 ' cells) Control Stimulated-[127.1 +2.8 129.5 i: 4.5 155.2_+2.9'
459.1 + I1.1 441. I + 8.4 406.5 + 7.4"
Monolayers of intact cells were washed 3 x 5 rain with 200/d/well of Pi-free gockes medium. The medium was replaced with 200 iH/well of treatment solution with (stimulated) or without (control) 20 l*M nicotine and incubated for l0 min at 37'C. Media were recovered at the end of the incubation for determination of secreted catecholamines. *Significantly different from other groups in the column at P < 0.05. +All stimulated groups are significantly different from their respective control groups ut P < 0.05. Each table value is the mean + SEM for 8 replicnles
Table 3. [3SS] thiophosphate uptake by chromaffin cells during secretion in media with varying phosphate and thiophosphate content
Treatment Normal medium P,-tYee medium 3 m M P~-medium m M TP~-medium
['~S] TP i uptake (dpm} Control Stimulaled 6583 + 122 54,356 +- 1523 6985 ± 309 1065 ! 4 ~
6938 q: [90 36,104 + 1269* 6492 4 315 1148 ! 20#
Monolayers of chromalfin cells (2 × 10 ~} were washed 3 times for 5 rain at 37 (7 with 200 /d/well of Lockes solution. The solution was replaced with 200 l*l/well of treatment solution containing 10 ltCi/ml of [~SS]TP, without (control) or with 20 f~M nicotinc (stimulated) and the cells incubated for [0 min at 37 . The monolayers were rapidly washed 4 times with 200 ,ul/well of ice cold treatment solutions free of either radioactivity or nicotine. The monolayers were then dissolved in 50 HI/well of SDS sample buffer (without mercaptoethanol) used for SDS P A G E and counted along with two 50/d washes. * Significantly different from the respective control group at P < 0.05. + Significantly different (except for the respective control group) from all other groups at P < 0.05.
groups. Uptake of [~SS]TP~ from 3 mM TP, medium was only about 16% that of either the normal or 3 mM P~ medium. Since only trace levels of TP~ were present in the P~-free medium, it is to be expected that radiolabel uptake would be greatest for these cells. However, uptake by stimulated cells in this medium was only about 65% that of unstimulated cells. Uptake of [3SS]TP~ from media indicated that the label should be incorporated into cellular proteins after conversion to ATPTS. Cells were cultured with media containing [35S]TP, for periods of 1-3 days. The medium was removed and the cells dissolved in SDS sample buffer for SDS-PAGE. As shown in Fig. IA, more 35S was incorporated into cell proteins in media containing P~ than in media containing TP,. A 10-fold increase in [35S]TPi for the group treated with TP~ medium (200 t~Ci/ml) resulted in a banding pattern on SDS PAGE that was similar to that seen with the other media (Fig. IA). Incorporation by ceils in P,-free medium was similar to that for cells cuhured in medium containing P~. Label appeared primarily in a broad 97 121 kDa protein band; 2-D electrophoresis of this sample is shown in Fig. lB. Thiophosphoproteins represented by the 97 121 kDa band on SDS-PAGE separated into two clusters near the neutral and acidic ends of the first dimension get. The 47 kDa protein which we have previously described (Brooks and Brooks, 1985) was not strongly thiophosphorylated under these experimental conditions.
Morpholoqic and metabofic t~ffect qf TP~ Cells were cultured in the media described in Table 1 for periods of 1-3 days and the effects of the media on the morphology of the cells evaluated by trans-
Thiophosphate in chromaffin cells kDa
--116 --97 --
67'
B43
pH 10
3.0
Fig. 1. Thiophosphate incorporation into cellular proteins. (A) Chromaffin cells were cultured for 3 days in the presence of various media containing 20 ~Ci/ml (200/~Ci/ml in Lane 6) of [35S]thiophosphate. Lanes 1 and 5, normal medium; Lane 2, P~-freemedium ; Lane 3, 3 mM Pi medium; Lane 4, 3 mM TP~ medium; and Lane 6, repeat of Lane 4 at 200 pCi/ml. The cell proteins were separated by SDS PAGE and the radiolabelled proteins visualized by fluorography. The molecular masses of the protein standards are indicated by the numbers to the right. (B) Samples of cells incubated with normal medium were subjected to 2-dimensional electrophoresis before fluorography. The arrows indicate the approximate molecular masses of the radiolabelled proteins present in the broad band on the single dimension SDSPAGE gel. mission electron microscopy. The appearance of cells cultured in normal medium, P~-free medium or 3 mM P~ medium (Fig. 2, panels 1-3, respectively) showed a normal appearance, similar to previous descriptions of cultured cells (Brooks and Carmichael, 1987). The appearance of cells cultured in 3 mM TP~ medium (Fig. 2, panel 4) showed the depletion of chromaffin vesicles which correlates with the analytical measurements of total cellular catecholamine content. There was also extensive damage to the mitochondria. The
515
mitochondria were swollen in many instances with almost complete disruption of the cristae after 3 days of culture with media containing 3 mM TP~ (Fig. 2, panel 4B). Damage to mitochondrial function should be reflected in reduced metabolic activity by the cells grown in medium containing TP~. The dye MTT is converted to its blue crystalline form MTT-formazan by metabolically active cells and has been used to assess viability, proliferation and cytotoxicity in cell cultures (Mosman, 1983). The modification of Magilavy and Rothstein (1988) was used in these studies as an assay for the general metabolic activity of cultured cells. Chromaffin cells were cultured in the four media indicated in Table 1 for periods of 1-3 days and their metabolic activity judged by MTT assay. The cells showed enhanced MTT conversion after one day in culture and a markedly reduced activity by the third day of culture (Table 4). Differences in dye accumulation by chromaffin cells could be observed microscopically within minutes of exposure to MTT. Neutral red uptake has been used to identify chromarlin cells in preparations of isolated cells since the dye is actively taken up by chromaffin vesicles (Role and Perlman, 1980). It has also been used to vitally stain catecholaminergic neurons (Stuart et al., 1974) and the secretory granules of juxtaglomerular cells (Fray et al., 1987). We used uptake of the dye by cells treated with various media to indicate the general quality of chromaffin vesicle function. At the end of 1-3 days of culture in the various media, the medium was replaced briefly with a solution of neutral red in saline. Dye uptake by cells cultured in medium containing TP~ was reduced by 33% compared to the other groups (Table 5). Differences in neutral red uptake by chromaffin cells in the different treatment groups were easily observed by microscopic examination of the cultures. The reliability of several of the analytical measurements requires that exposure to the media does not result in the loss of viable cells from the culture plates. D N A measurements were made of cells cultured in the various media for the times used in the experiments. There was no significant difference (data not shown) in the D N A content for cultures carried in parallel through any of the experiments, demonstrating that the results do not reflect cell destruction or physical loss from the culture substrata, DISCUSSION
Chromaffin cells were incubated with TP~ using a treatment protocol analogous to that of ortho-
(3B~
~4 Fig. 2. Transmission electron microscopy of chromattin cells in various media. Chromaffin cells were cultured in various media for 3 days on Lux T h e r m a n o x membranes. Samples were fixed and stained for transmission electron microscopy. For each pair of panels, the left is low magnification (4100 x ) and the right is high magnification (9200x). I A,B, normal medium; 2 A,B, Pi-free medium; 3 A,B, 3 m M P, medium ; and, 4 A,B, 3 m M T E medium. The arrowheads indicate damaged mitochondria. 516
Thiophosphate in chromaffin cells Table 4. MTT formazan formation by chromaffin cells cultured in various media Treatment medium Normal medium Pi-free medium 3 mM Pimedium 3 mM TPi medium
#mol MTT formazan/2 x 105 cells Day 1 Day 2 Day 3 0.239+_0.005 0.299_+0.004 0.293+_0.003 0.341+0.004"
0.265+_0.004 0.287+_0.004 0.259+_0.008 0.247-+0.008
0.225+_0.016 0.343+_0.002 0.326_+0.014 0.168+0.003~-
Cells were cultured with 0.2 ml/wen of the indicated media for periods of 1-3 days. After the culture period, 0.1 ml/well of medium (including medium-only blanks) was removed and 20 #1 of MTT solution added. The plates were returned to the incubator for 1 h. The reaction was terminated by the addition to each well of 0.1 ml of 10% SDS in 0.01 M HCI. The plates were returned to the incubator overnight to dissolve the formazan crystals. The absorbance of the individual wells was determined at 570 nm and converted to #mol MMT formazan. * Significantlydifferent from the values for the other treatment groups on day 1 at P < 0.05. ~'Significantlydifferent from the other values in this column as well as from the day I and 2 values for this treatment group at P < 0.05. Each table value is the mean _+SEM for 4 replicates. Table 5. Neutral red uptake by chromattin cells incubated for 3 days in media containing phosphate or thiophosphate Treatment Normal medium Prfree medium 3 mM Pi-medium 3 mM TPi-medium
Neutral red uptake (ng/2 x 105 cells) 20.5 +-0.1 20.5 + 0.3 21.5+0.2" 13.8 + 0.3*
Chromaffin cells were cultured for 3 days in normal medium, P~-free medium, or P~-freemedium supplemented with 3 mM P~or TP~. At the end of the incubation period, the medium was removed, the cells washed once with 200 #l/well of saline and exposed for 10 min to 200 #l/well of 0.6 mg/ml neutral red in saline. The cells were washed 4 times with 200 #l]well of saline and the cell monolayer dissoved in 200 #l/well of 1% SDS in 0.01 M HCI. An aliquot was diluted to 1.5 ml with the same solution and the dye concentration of the cells determined fluorometrically. All manipulations of cells were carried out at 3T'C. Each table value is the mean + SEM for 6 replicates. *Different from all other groups at P < 0.05.
p h o s p h a t e in protein p h o s p h o r y l a t i o n experiments. Unlike P~, TP~ caused severe d a m a g e to the secretory system o f cultured chromaffin cells. The cells lost 75 % o f their catecholamines over a 3 - 4 day period. This was associated with a progressive decline in b o t h absolute catecholamine secretion a n d the ratio of s t i m u l a t e d / u n s t i m u l a t e d secretion, until the cells became unresponsive to stimulation. T h e m e c h a n i s m o f cell c a t e c h o l a m i n e loss is p r o b a b l y depletion without replacement, since even a brief exposure to TP~ caused a n increase in basal catecholamine release. C o m p a r i s o n o f the D N A c o n t e n t for cultures treated with the various media showed t h a t the changes seen could n o t be explained as a consequence o f cell death or loss from the culture substrata during either
517
m e d i u m removal or washes. R e t u r n of the cells to n o r m a l m e d i u m did n o t restore either their catec h o l a m i n e c o n t e n t or ability to secrete, indicating t h a t TPi has a p e r m a n e n t effect o n cell function. T h e u p t a k e o f [3SS]TPi into cultured cells d u r i n g a 10 m i n assay period d e m o n s t r a t e s t h a t the cells are capable o f rapid TP~ uptake. However, it is n o t k n o w n if TP~ is t a k e n up by the same m e c h a n i s m t h a t is n o r m a l l y involved in P~ u p t a k e or if it m i g h t be influenced by changes in t r a n s m e m b r a n e Pi levels which occur during secretion. It seems unlikely t h a t in such a s h o r t period the effect could be attributed to TP~induced d a m a g e to cell metabolism since stimulated secretion is only slightly reduced. However, transcellular P~ levels could change e n o u g h d u r i n g secretion to competitively inhibit TP~ t r a n s p o r t or enzyme reactions. This m a y explain the results with P
019%0186/9255.00+0.00 Copyright © 1992 Pergamon Press Ltd
INORGANIC THIOPHOSPHATE EFFECTS ON CHROMAFFIN CELL STRUCTURE AND FUNCTION JACK C. BROOKS,I* MELINDA H. BROOKSI and STEPHEN W. CARMICHAEL2 LMarquette University School of Dentistry, Department of Basic Sciences, 604 North 16th Street, Milwaukee, WI 53233, U.S.A. 2Department of Anatomy, Mayo Clinic, Medical Science Bldg. 3, Rochester, MN 55905, U.S.A. (Received 20 May 1991 ; accepted 9 October 1991)
Abstract--The effect was determined of replacing medium inorganic phosphate with thiophosphate on the structure and function of cultured bovine chromaffin cells. Cell cultures were incubated in normal medium containing fetal bovine serum, phosphate free medium or similar medium supplemented with inorganic phosphate or thiophosphate. In contrast to the other media, cells cultured with thiophosphate medium for 3~, days showed seriously compromised structure and functions. The cells lost 75% of their catecholamine content and their ability to secrete remaining catecholamines in response to nicotine stimulation. Radiolabelled thiophosphate was rapidly taken up by the cells and, in long-term experiments, was incorporated largely into a 97 121 kDa protein band on SDS-PAGE. Additional minor bands were found to a lesser, variable extent. Transmission electron micrograpbs of cells treated with thiophosphate showed extensive depletion of chromaffin vesicles and disruption of mitochondria, suggesting that the functional damage noted with these cells could be associated with damage to mitochondria. Analysis of general cell metabolic activity by conversion of the dye (3-[3,4-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide) to its formazan derivative indicated increased metabolic activity at early stages of exposure to thiophosphate followed by a decline with continued exposure, supporting the argument for an overall depression of cell metabolism. Uptake of the dye neutral red, which is avidly accumulated by chromaffin cells, was also reduced for cells exposed to thiophosphate. The data suggest that thiophosphate enters chromaffin cells and disrupts energy dependent cell functions, including catecholamine storage and secretion.
N u m e r o u s studies have suggested a role for protein phosphorylation in the process of catecholamine secretion from chromaffin cells (Amy and Kirshner, 1981 ; Cote et al., 1986 ; Lee and Holz, 1986 ; Haycock et al., 1988a,b; Brooks and Brooks, 1987a,b; Burgoyne, 1984). Many of these experiments share the c o m m o n protocol of incubating the cultured cells with 32Pi to permit them to incorporate it into ATP. The [7-32p]ATP is then available as substrate for protein phosphorylation reactions and these reactions are correlated with elicited cellular responses. Numerous proteins are phosphorylated in chromaffin cells, in association with secretion, in either intact or permeabilized cells. A recent study (Gutierrez et al., 1988) used this protocol with two-dimensional electro-
phoresis to map over 500 phosphoproteins in chromaffin cells. The A T P analog ATPTS (adenosine-5'-O-(3-thiotriphosphate)) also serves as a substrate for (thio)phosphorylation reactions. However, protein thiophosphorylation reactions are generally considered irreversible because cellular phosphatases remove the thiophosphoryl groups very slowly or not at all (Cassidy et al., 1979; Sherry et al., 1978; Coyne et al., 1987). Our strategy has been to treat permeabilized chromaffin cells with ATPTS to lock phosphorylation reactions in the thiophosphorylated state. This then permits us to evaluate the relationship between such reactions and the secretory process. Secretion by permeabilized cells is inhibited by exposure to ATPTS and is correlated with the thiophosphorylation of several proteins (Brooks and Brooks, 1985, 1987a,b). The data have led us to the hypothesis that a phos*Author to whom correspondence should be addressed. phorylation reaction may be important in the control Abbreviations: MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diof secretion, although a causal relationship has not phenyltetrazolium bromide) ; MTT formazan, (l-[4,5dimethylthiazole-2-yl]-3,5-diphenylformazan), TP~, thio- been established. In the experiments reported here, we have treated phosphate ; ATP?S, adenosine-5'-O- (3-thiotriphosphate). intact chromaffin cells with inorganic thiophosphate 511
JACK C. BROOKS el al.
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using a p r o t o c o l a n a l o g o u s to t h a t used in e x p e r i m e n t s with i n o r g a n i c p h o s p h a t e . T h e objective was to force the cells to synthesize ATPTS so that p h o s p h o r y l a t i o n reactions c o u l d be s t u d i e d in intact cells. W e h y p o t h e s i z e d t h a t the t h i o p h o s p h a t e w o u l d be t a k e n up by the cells a n d synthesized into ATPTS, w h i c h w o u l d t h e n be used for p r o t e i n p h o s p h o r y l a t i o n reactions. T h e e x p e c t a t i o n was t h a t we could devise specific t r e a t m e n t r e g i m e n s w h i c h w o u l d allow us to identify the role o f various p h o s p h o r y l a t i o n reactions in cell functions. In particular, p r o t e i n s essential to secretion m i g h t be p e r m a n e n t l y t h i o p h o s p h o r y l a t e d a n d affect the cell secretory response.
EXPERIMENTAL PROCEDURES
Materials All reagents were analytical grade or the best available grade. Dulbecco's modified Eagle Medium, HAM F-12 Nutrient Mixture and antibiotics were purchased from GIBCO (Grand Island, NY). Dulbecco's Modified Eagle's Medium (High glucose, phosphate-free, HEPES-buffered) was obtained from Irvine Scientific (Santa Ana, CA) as a powder, prepared and filter sterilized before use. This medium was designated as P~-free medium; it was supplemented with 3 mM Na3PO4 (3 mM Pi-medium) or 3 mM Na3PO3S" 12H20 (3 mM TPi-medium) for cell culture. The initial pH of all media was adjusted to 7.4. Carrier free sodium thiophosphate, specific activity 48.1 TBq/mmol, was purchased from New England Nuclear lnc., Boston, MA. Nicotine, MTT, MTT formazan, neutral red (purified), Hoechst 33258 (bisbenzamide) and calf thymus DNA were obtained from Sigma Chemical Co., St Louis, MO. Tris (gold label) was obtained from Aldrich Chem. Co., Milwaukee, W1. An electrophoresis calibration kit for low molecular weight proteins was obtained from Pharmacia (Piscataway, N J). Other electrophoresis chemicals were obtained from Polysciences Inc., Warren, PA. General experimental protocol Bovine chromaffin cells were prepared and maintained in primary culture as described previously (Brooks and Treml, 1983). Isolated cells were suspended at 106 cells/ml and plated at 2 x 10 s cells/well in 96 well culture plates (Falcon, 3070) or, for experiments involving protein thiophosphorylation, at 106 cells/well in 24 well plates (Corning, Cell Wells 25820). The medium was changed after 4 days and the cells were used between 4 and 8 days of culture. Cytosine arabinoside was used at 10/tM concentration in the culture medium to inhibit fibroblast growth. The cultures contained about 85% chromaffin cells. Celt monolayers used for experiments were washed 3 x 5 min. with 0.2 ml/well of complete Lockes solution (Brooks and Treml, 1983), 0.9% saline or Pi-free medium, as required by the experiment. The cells were then exposed to 0.2 ml/well of treatment solutions. In some experiments Lockes solution was prepared without phosphate and supplemented with either 3 mM P, or TP~. This was used to determine the effect of TP~ presence in the medium during stimulated secretion. Secretion was elicited with 20 itM nicotine. MTT was solu-
bilized in Tris buffered saline (50 mM Tris CI. 0.145 M NaCI, pH 7.4).
Analytical methods Total cellular catecholamine content and catecholamine released to the medium during assay of secretion was determined fluorometrically as described previously (Brooks and Treml, 1983). The catecholamine content of individual samples was determined from a standard curve relating relative fluorescence to known concentrations of a 1:I mixture of norepinephrine and epinephrine. For determination of total cellular catecholamine content, cell monolayers were dissolved in 0.2 ml/well of 0.01 M HC1. Data are expressed as ng catecholamine/2 x 105 cells as indicated in the tables. Since high concentrations of thiophosphate interfered slightly with the fluorometric assay, a standard curve was constructed in the presence of 3 mM TP~ for measurements of secretion made in media containing thiophosphate. Total DNA conlent of monolayers was used to determine if experimental conditions resulted in the loss of viable cells. The method used was that of Cesarone et al. (1979), using calf thymus DNA as a standard. DNA standards and monolayers were dissolved in the specified citrate buffer containing 1% SDS (100 #1/well, plus one 100 #1 wash) so that the final concentration of SDS in the assay mixture did not exceed 0.05 %. A fluorometric assay based upon the intrinsic fluorescence of neutral red was designed for the determination of neutral red accumulation by cells. The excitation and emission wavelengths used were 510 and 600 nm, respectively. A standard curve was constructed using relative fluorescence against known concentrations of neutral red in a solution containing t% SDS and 0.01 M HCI; the curve was linear over the range of 0.5 25 ng neutral red/ml. For assay of neutral red accumulation, monolayers of cells incubated with various trcatmem media were washed quickly three times with 0.2 ml/well of 0.9% saline at 3 7 C and incubated for 10 rain with a 0.6 mg/ml solution of neutral red in saline. The cells were then washed four times with 0.2 ml/well of saline and dissolved in 1% SDS in 0.0l M HC1 for determination of neutral red accumulation. The conversion of MTT to MTT formazan was used to determine the metabolic effect and possible cytotoxicity of the different media. The metabolic activity of cells cultured for periods of l 3 days with various media was assayed as described by Magilavy and Rothstein (1988). At the end of the culture period, 100 #1 of medium was removed from each well, 20 #1 of MTT solution (5 mg MTT/ml TBS) was added and the plates returned to the incubator for 1 h. The cells were dissolved at the end of this period by addition of 100 #1/well of 10% SDS in 0.01 M HCI. The plates were returned to the incubator overnight to dissolve the blue MTT formazan reaction product. The formazan was quantitated spectrophotometrically at 570 nm (690 nm reference) on a Minireader-ll microelisa reader (Dynatech Laboratories Inc., Alexandria, VA) against a standard curve relating A , ~ to concentration of MTT formazan. Medium (without cells) was carried through all culture periods to serve as blanks for the assays. Statistical analysis was performed by one-way ANOVA on a VAX system using SPSSX (SPSS Inc.. Chicago, IL) at a significance level of P < 0.05. Determination q/thiophosphate uptake and protein thiophospharvlation For experiments involving the determination of [~S]TP~ uptake by cells, treatment solutions were removed and the
Thiophosphate in chromaffin cells monolayers washed quickly four times with 0.2 ml/well of ice-cold treatment solutions similar to those used in the experiment but free of [35S]TP~. The cells were dissolved in 50/A/well of SDS sample buffer (without mercaptoethanol) used for SDS-PAGE, combined with two similar washes and counted in 7 ml of Aquasol-2 (New England Nuclear Inc., Boston, MA) as previously described (Brooks and Brooks, 1987a). Thiophosphorylation of cellular proteins was determined by [3SS]TPi incorporation into proteins from P~-free medium or media containing P~ or TPI and 20 #Ci/ml of [35S]TPi. After removal of the medium, the monolayers were dissolved directly in sample buffer and subjected to SDSPAGE in 10% polyacrylamide gels (Brooks and Brooks, 1985). Two-dimensional electrophoresis was carried out as described by Hochstrasser et al. (1988). The second dimension SDS-PAGE gels were stained with colloidal Coomassie brilliant blue G-250 (Neuhoff et al., 1988). Proteins thiophosphorylated with [35S]TPi were detected by fluorography for 2-8 day exposures at - 7 0 ° C on Kodak XOmat AR film after treatment of the Coomassie blue stained gel with sodium salicylate (Chamberlain, 1979). Gels were run in a Bio-Rad Mini-Protean system using gel and buffer formulations (SDS-PAGE) provided with the apparatus. The molecular weights of the separated proteins were determined from a curve relating the log molecular weight to migration distance for proteins of known molecular weight. Electron microscopy Cells cultured with the various media were subjected to transmission electron microscopy as previously described (Brooks and Carmichael, 1987). The ceils were cultured for 1 3 days on Lux Thermanox membranes (Miles Laboratories, Naperville, IL) in Micro slide chambers (Bellco Glass, Vineland, N J). The medium was removed and the cells (on the Lux membrane) placed in a freshly prepared solution of 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 at room temperature and transferred to a cold room at 4°C for 1 h. The cells were then washed briefly three times with icecold 0.3 M sucrose buffered with 0.I M cacodylate buffer, pH 7.2. The cells were treated with 1% osmium tetroxide for 1 h, dehydrated, and embedded. Sections were cut on a plane perpendicular to the Lux membrane surface, mounted on copper grids and stained with uranyl acetate and lead citrate. The specimens were examined with a Philips CM 12 electron microscope at 60 kV accelerating voltage.
RESULTS T P i d a m a g e to c a t e c h o l a m i n e content a n d secretion
A n initial m e d i u m concentration o f TP~ was chosen arbitrarily as 3 m M ; this is a b o u t three times the normal medium P~ concentration. The effect o f chronic exposure o f the cells to normal m e d i u m (F12, Dulbecco's Modified Eagle's Medium, 10% fetal bovine s e r u m ; see Brooks and Treml, 1983), P~-free medium and media supplemented with either 3 m M P~ or TP~ is shown in Table 1. N o r m a l medium is that customarily used for primary culture o f chromaffin cells and was included as a reference to which all other media were c o m p a r e d for normal cell function. The P~-free m e d i u m was included as a reference for the
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Table 1. Effectof chronic exposureof chromaffincells to elevatedP~ or TP~on cellcatecholaminecontent and secretion Exposure time
Catecholaminesecretionand cellularcontent (ng/2 x 105 cells) Normal Pl-free 3 mM Pi 3 mM TP~
Day 1"
128.0+6.5 123.3+__7.1 117.5+4.1 55.9 + 2,2:~ 372.2_+12.9 345.8+_8.4 336.4+_7.6 138.3_+8.2 3618_+56 3244+64 3348_+113 1392±50t
Day3
159.9-+4.2 147.9_+5.1 169.7_+3.2 45.7_+2.2:~ 620.0_+21.9 574.8_+6.4 505.0_+13.7 92.3_+9.6 3778_+80 3585_+104 4070+-135 1013_+54"I"
Day 4
255.7+47.3 249.6_+4.2 234.2_+9,0 65.1_+4.5~ 635.8+-17.8 775.1_+20.6 613.1+-15.1 76.0+_6.7 4464_+115 4460_+146 4072_+132 1077_+55"t
Chromaffin cells were cultured in normal medium for 4 days after isolation. The monolayerswere then exposed to 200 #l/well of the treatment solutions and incubated for the periods indicated in the table. For assay, the monolayerswere washed 3 × 5 min with 200 /~l/well of complete Lockes solution at 37°C. Total cellular catecholaminecontent was determined on aliquots of cellsdissolvedin 0.01 M HCI. Secretionwas assayedby exposing cellsto 200/ll/wellof Lockessolution(control)or Lockessolution +20/~M nicotine (stimulated) for 10 min at 37°C. The medium was recoveredand an aliquot taken for determinationof secreted catecholamines. *Under each treatment period, the values listed are unstimulated secretion, stimulated secretion and total cellular catecholamine content. Each table value is the mean+ SEM for 6 (secretion)or 8 (total catecholamine)replicates. ]'Significantly decreased compared to other total catecholamine groups at P < 0.05. :~Significantlydecreasedcompared to other unstimulated groups at P < 0.05. For day 4, the respectivestimulatedgroup is not different from the unstimulatedgroup. effect o f exogenous P~ and TP~ o n cell behavior. Total cellular catecholamine content was reduced by nearly 40% after one day o f exposure to TP~ medium. The absolute magnitude o f catecholamine secretion was similarly reduced, although the ratio o f stimulated to unstimulated release was not different from that o f cells exposed to any o f the other media. After 3 days o f culture the total cellular catecholamine content was reduced to about 25% that o f the other treatment groups and remained at that level through day 4 o f culture. Total secretion was markedly reduced by day 3 o f culture and stimulated secretion was lost by day 4 o f culture even though the cell catecholamine content had stabilized at a b o u t 25% o f normal. The effects o f chronic exposure to elevated TP~ suggested that reduced concentrations might have a similar effect and that perhaps the cells could recover from TP~-induced damage if they were returned to normal medium after a 3 day exposure to TPi medium. Cells were exposed (data not shown) to normal, P~-free medium or medium supplemented with 1-3 m M P~ or TP~ for 3 days. TP~ at 1 m M concentration had no significant effect on either total cellular catecholamine content or secretion over the 3 day exposure period,
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JACK C. BROOKS el ~ll.
while 2 and 3 mM TP~ significantly reduced total cellular catecholamine content and secretion. Cells exposed to those media for 3 days were returned to normal medium for an additional 3 day period to determine if the TP,-treated cells would recover both their normal catecholamine content and secretory responsiveness. The catecholamine content of all groups remained the same as it was before the cells were returned to normal medium. The cells originally cultured with TP, medium showed no significant improvement in total cellular catecholamine content or stimulated secretion upon return to normal medium. Exposure of cells to 3 mM TP~ in Lockes solution for a 2 h period had no effect on the subsequent secretory responsiveness of the cells in Lockes solution free of TPj. However, stimulated secretion was significantly reduced when the cells were pretreated for 2 h with either 3 mM TPi or P~ in Lockes solution but then stimulated by 20 JiM nicotine in the presence of TP, (not shown). This result suggested that TP~ might have a direct inhibitory effect on secretion and might influence secretion simply by being present during the usual 10 min assay period. The presence of 3 mM TP~ in the assay medium caused a slight reduction of stimulated secretion (Table 2) and a slight increase in unstimulated release when compared to 3 mM P~ medium. The slight inhibitory effect of TPL when present during the assay of secretion suggested that TP~ might enter the cell and affect secretion. If true, this should be reflected in altered uptake of [3SS]TP~ from media. Data describing the uptake of [-~SS]TP, (10 tlCi/ml) from various media is shown in Table 3. Uptake of radiolabel was similar for normal and 3 mM P~ medium for respective unstimulated and stimulated Table 2. Effect of varying phosphale and thiophosphate on catecholamine secretion
Treatmenl P j r c e Lockes 3 m M P,-Lockes 3 m M TPi-Lockes
Catecholamine secretion (ng/2 × 10 ' cells) Control Stimulated-[127.1 +2.8 129.5 i: 4.5 155.2_+2.9'
459.1 + I1.1 441. I + 8.4 406.5 + 7.4"
Monolayers of intact cells were washed 3 x 5 rain with 200/d/well of Pi-free gockes medium. The medium was replaced with 200 iH/well of treatment solution with (stimulated) or without (control) 20 l*M nicotine and incubated for l0 min at 37'C. Media were recovered at the end of the incubation for determination of secreted catecholamines. *Significantly different from other groups in the column at P < 0.05. +All stimulated groups are significantly different from their respective control groups ut P < 0.05. Each table value is the mean + SEM for 8 replicnles
Table 3. [3SS] thiophosphate uptake by chromaffin cells during secretion in media with varying phosphate and thiophosphate content
Treatment Normal medium P,-tYee medium 3 m M P~-medium m M TP~-medium
['~S] TP i uptake (dpm} Control Stimulaled 6583 + 122 54,356 +- 1523 6985 ± 309 1065 ! 4 ~
6938 q: [90 36,104 + 1269* 6492 4 315 1148 ! 20#
Monolayers of chromalfin cells (2 × 10 ~} were washed 3 times for 5 rain at 37 (7 with 200 /d/well of Lockes solution. The solution was replaced with 200 l*l/well of treatment solution containing 10 ltCi/ml of [~SS]TP, without (control) or with 20 f~M nicotinc (stimulated) and the cells incubated for [0 min at 37 . The monolayers were rapidly washed 4 times with 200 ,ul/well of ice cold treatment solutions free of either radioactivity or nicotine. The monolayers were then dissolved in 50 HI/well of SDS sample buffer (without mercaptoethanol) used for SDS P A G E and counted along with two 50/d washes. * Significantly different from the respective control group at P < 0.05. + Significantly different (except for the respective control group) from all other groups at P < 0.05.
groups. Uptake of [~SS]TP~ from 3 mM TP, medium was only about 16% that of either the normal or 3 mM P~ medium. Since only trace levels of TP~ were present in the P~-free medium, it is to be expected that radiolabel uptake would be greatest for these cells. However, uptake by stimulated cells in this medium was only about 65% that of unstimulated cells. Uptake of [3SS]TP~ from media indicated that the label should be incorporated into cellular proteins after conversion to ATPTS. Cells were cultured with media containing [35S]TP, for periods of 1-3 days. The medium was removed and the cells dissolved in SDS sample buffer for SDS-PAGE. As shown in Fig. IA, more 35S was incorporated into cell proteins in media containing P~ than in media containing TP,. A 10-fold increase in [35S]TPi for the group treated with TP~ medium (200 t~Ci/ml) resulted in a banding pattern on SDS PAGE that was similar to that seen with the other media (Fig. IA). Incorporation by ceils in P,-free medium was similar to that for cells cuhured in medium containing P~. Label appeared primarily in a broad 97 121 kDa protein band; 2-D electrophoresis of this sample is shown in Fig. lB. Thiophosphoproteins represented by the 97 121 kDa band on SDS-PAGE separated into two clusters near the neutral and acidic ends of the first dimension get. The 47 kDa protein which we have previously described (Brooks and Brooks, 1985) was not strongly thiophosphorylated under these experimental conditions.
Morpholoqic and metabofic t~ffect qf TP~ Cells were cultured in the media described in Table 1 for periods of 1-3 days and the effects of the media on the morphology of the cells evaluated by trans-
Thiophosphate in chromaffin cells kDa
--116 --97 --
67'
B43
pH 10
3.0
Fig. 1. Thiophosphate incorporation into cellular proteins. (A) Chromaffin cells were cultured for 3 days in the presence of various media containing 20 ~Ci/ml (200/~Ci/ml in Lane 6) of [35S]thiophosphate. Lanes 1 and 5, normal medium; Lane 2, P~-freemedium ; Lane 3, 3 mM Pi medium; Lane 4, 3 mM TP~ medium; and Lane 6, repeat of Lane 4 at 200 pCi/ml. The cell proteins were separated by SDS PAGE and the radiolabelled proteins visualized by fluorography. The molecular masses of the protein standards are indicated by the numbers to the right. (B) Samples of cells incubated with normal medium were subjected to 2-dimensional electrophoresis before fluorography. The arrows indicate the approximate molecular masses of the radiolabelled proteins present in the broad band on the single dimension SDSPAGE gel. mission electron microscopy. The appearance of cells cultured in normal medium, P~-free medium or 3 mM P~ medium (Fig. 2, panels 1-3, respectively) showed a normal appearance, similar to previous descriptions of cultured cells (Brooks and Carmichael, 1987). The appearance of cells cultured in 3 mM TP~ medium (Fig. 2, panel 4) showed the depletion of chromaffin vesicles which correlates with the analytical measurements of total cellular catecholamine content. There was also extensive damage to the mitochondria. The
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mitochondria were swollen in many instances with almost complete disruption of the cristae after 3 days of culture with media containing 3 mM TP~ (Fig. 2, panel 4B). Damage to mitochondrial function should be reflected in reduced metabolic activity by the cells grown in medium containing TP~. The dye MTT is converted to its blue crystalline form MTT-formazan by metabolically active cells and has been used to assess viability, proliferation and cytotoxicity in cell cultures (Mosman, 1983). The modification of Magilavy and Rothstein (1988) was used in these studies as an assay for the general metabolic activity of cultured cells. Chromaffin cells were cultured in the four media indicated in Table 1 for periods of 1-3 days and their metabolic activity judged by MTT assay. The cells showed enhanced MTT conversion after one day in culture and a markedly reduced activity by the third day of culture (Table 4). Differences in dye accumulation by chromaffin cells could be observed microscopically within minutes of exposure to MTT. Neutral red uptake has been used to identify chromarlin cells in preparations of isolated cells since the dye is actively taken up by chromaffin vesicles (Role and Perlman, 1980). It has also been used to vitally stain catecholaminergic neurons (Stuart et al., 1974) and the secretory granules of juxtaglomerular cells (Fray et al., 1987). We used uptake of the dye by cells treated with various media to indicate the general quality of chromaffin vesicle function. At the end of 1-3 days of culture in the various media, the medium was replaced briefly with a solution of neutral red in saline. Dye uptake by cells cultured in medium containing TP~ was reduced by 33% compared to the other groups (Table 5). Differences in neutral red uptake by chromaffin cells in the different treatment groups were easily observed by microscopic examination of the cultures. The reliability of several of the analytical measurements requires that exposure to the media does not result in the loss of viable cells from the culture plates. D N A measurements were made of cells cultured in the various media for the times used in the experiments. There was no significant difference (data not shown) in the D N A content for cultures carried in parallel through any of the experiments, demonstrating that the results do not reflect cell destruction or physical loss from the culture substrata, DISCUSSION
Chromaffin cells were incubated with TP~ using a treatment protocol analogous to that of ortho-
(3B~
~4 Fig. 2. Transmission electron microscopy of chromattin cells in various media. Chromaffin cells were cultured in various media for 3 days on Lux T h e r m a n o x membranes. Samples were fixed and stained for transmission electron microscopy. For each pair of panels, the left is low magnification (4100 x ) and the right is high magnification (9200x). I A,B, normal medium; 2 A,B, Pi-free medium; 3 A,B, 3 m M P, medium ; and, 4 A,B, 3 m M T E medium. The arrowheads indicate damaged mitochondria. 516
Thiophosphate in chromaffin cells Table 4. MTT formazan formation by chromaffin cells cultured in various media Treatment medium Normal medium Pi-free medium 3 mM Pimedium 3 mM TPi medium
#mol MTT formazan/2 x 105 cells Day 1 Day 2 Day 3 0.239+_0.005 0.299_+0.004 0.293+_0.003 0.341+0.004"
0.265+_0.004 0.287+_0.004 0.259+_0.008 0.247-+0.008
0.225+_0.016 0.343+_0.002 0.326_+0.014 0.168+0.003~-
Cells were cultured with 0.2 ml/wen of the indicated media for periods of 1-3 days. After the culture period, 0.1 ml/well of medium (including medium-only blanks) was removed and 20 #1 of MTT solution added. The plates were returned to the incubator for 1 h. The reaction was terminated by the addition to each well of 0.1 ml of 10% SDS in 0.01 M HCI. The plates were returned to the incubator overnight to dissolve the formazan crystals. The absorbance of the individual wells was determined at 570 nm and converted to #mol MMT formazan. * Significantlydifferent from the values for the other treatment groups on day 1 at P < 0.05. ~'Significantlydifferent from the other values in this column as well as from the day I and 2 values for this treatment group at P < 0.05. Each table value is the mean _+SEM for 4 replicates. Table 5. Neutral red uptake by chromattin cells incubated for 3 days in media containing phosphate or thiophosphate Treatment Normal medium Prfree medium 3 mM Pi-medium 3 mM TPi-medium
Neutral red uptake (ng/2 x 105 cells) 20.5 +-0.1 20.5 + 0.3 21.5+0.2" 13.8 + 0.3*
Chromaffin cells were cultured for 3 days in normal medium, P~-free medium, or P~-freemedium supplemented with 3 mM P~or TP~. At the end of the incubation period, the medium was removed, the cells washed once with 200 #l/well of saline and exposed for 10 min to 200 #l/well of 0.6 mg/ml neutral red in saline. The cells were washed 4 times with 200 #l]well of saline and the cell monolayer dissoved in 200 #l/well of 1% SDS in 0.01 M HCI. An aliquot was diluted to 1.5 ml with the same solution and the dye concentration of the cells determined fluorometrically. All manipulations of cells were carried out at 3T'C. Each table value is the mean + SEM for 6 replicates. *Different from all other groups at P < 0.05.
p h o s p h a t e in protein p h o s p h o r y l a t i o n experiments. Unlike P~, TP~ caused severe d a m a g e to the secretory system o f cultured chromaffin cells. The cells lost 75 % o f their catecholamines over a 3 - 4 day period. This was associated with a progressive decline in b o t h absolute catecholamine secretion a n d the ratio of s t i m u l a t e d / u n s t i m u l a t e d secretion, until the cells became unresponsive to stimulation. T h e m e c h a n i s m o f cell c a t e c h o l a m i n e loss is p r o b a b l y depletion without replacement, since even a brief exposure to TP~ caused a n increase in basal catecholamine release. C o m p a r i s o n o f the D N A c o n t e n t for cultures treated with the various media showed t h a t the changes seen could n o t be explained as a consequence o f cell death or loss from the culture substrata during either
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m e d i u m removal or washes. R e t u r n of the cells to n o r m a l m e d i u m did n o t restore either their catec h o l a m i n e c o n t e n t or ability to secrete, indicating t h a t TPi has a p e r m a n e n t effect o n cell function. T h e u p t a k e o f [3SS]TPi into cultured cells d u r i n g a 10 m i n assay period d e m o n s t r a t e s t h a t the cells are capable o f rapid TP~ uptake. However, it is n o t k n o w n if TP~ is t a k e n up by the same m e c h a n i s m t h a t is n o r m a l l y involved in P~ u p t a k e or if it m i g h t be influenced by changes in t r a n s m e m b r a n e Pi levels which occur during secretion. It seems unlikely t h a t in such a s h o r t period the effect could be attributed to TP~induced d a m a g e to cell metabolism since stimulated secretion is only slightly reduced. However, transcellular P~ levels could change e n o u g h d u r i n g secretion to competitively inhibit TP~ t r a n s p o r t or enzyme reactions. This m a y explain the results with P
