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Comparison of Dopamine β-hydroxylase activity in normal rat adrenal medulla and rat pheochromocytoma cells
144
Brain Research, 358 (1985) 144-149 Elsevier
BRE 11185
Comparison of Dopamine fl-Hydroxylase Activity in Normal Rat Adrenal Medulla and Rat Pheochromocytoma Cells DONA L. WONG and ROLAND D. CIARANELLO
Laboratory of Developmental Neurochemistry, Departmentof Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.) (Accepted February 25th, 1985)
Key words: rat pheochromocytoma cell - - dopamine fl-hydroxylase activity
Dopamine fl-hydroxylase activity is reportedly negligible in malignant rat adrenal cell lines. However, in two pheochromocytoma cell lines, PC12 and PCG2, considerable amounts of this catecholamine enzyme exist but its characteristics differ from the enzyme found in normal rat tissue in two ways. First, in normal adrenal medullary tissue, dopamine fl-hydroxylase activity increases linearly with increasing protein concentration. Second, there is a stringent requirement for copper. Concentrations of copper above or below the optimum inhibit enzymatic activity. In contrast, in pheochromocytoma ceils, dopamine fl-hy&oxytase exhibits a sigmoidal response with increasing tissue content. At dilute protein concentrations where considerable dopamine fl-hydroxylase activity is observed in normal adrenal medullary extracts, enzymatic activity is negligible in the p h e o e h r o ~ o m a cell lines. As the protein concentration is increased, activation of the enzyme occurs, and enzymatic activity increases linearly with further increases in protein concentration. At very high concentrations of protein, enzymatic activity plateaus. For both PC12 and PCG2 cells, doparaine p-hydroxylase activity shows minimal copper dependency, suggesting that endogenous inhibitors present in normal tissue are absent in these malignant cell lines. Under conditions of maximum activation, the activity of the enzyme in PC12 ceils becomes equivalent to that in normal rat adrenal medulla but remains 45-fold greater than that in PCG2 cells.
INTRODUCTION
(PNMT) activity are elevated but no changes in C A T occur.
The pheochromocytoma clonal cell lines, PC129 and PCG28, were derived from a rat adrenal medullary tumor originally defined by Warren and Chute t7. Both cell lines hypersecrete catecholamines, but appear to be regulated differently. For example, cultured PC12 cells divide but do not form processes. In the presence of nerve growth factor (NGF), cell division ceases, and neurite outgrowth begins. Concomitantly, choline acetyltransferase (CAT) activity is induced. If the PC12 cells are simultaneously supplemented with glucocorticoids, the NGF-stimulated induction of C A T is blocked. However, tyrosine hydroxylase activity is stimulated, The latter is in contrast to the effect of glucocorticoids in normal chromaffin tissue where both tyrosine hydroxylase (TH) and phenylethanolamine N-methyltrausferase
Although derived from the same malignant tissue, cultured PCG2 cells behave oppositely 8. N G F does not induce the changes in morphology and growth rate in the P C G 2 cell line as observed in the PC12 cell line. Neurite outgrowth is not stimulated, but tyrosine hydroxylase induction occurs at levels comparable in magnitude to that seen in intact animals ~5. Dexamethasone potentiates this NGF-induced increase in TH in P C G 2 cells as it does in normal sympathetic neurons. In general, PCG2 cells appear to more closely mimic the response to growth factors and steroids seen in norma! sympathetic neurons, both inthe intact animal or grown in organ culture. Previous studies have suggested that dopmafine flhydroxylase ( D B H ) , the enzyme responsible for norepinephrine synthesis, is either absent or present in
Correspondence: D.L. Wong, Laboratory of Developmental Neurochemistry, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
145 negligible amounts in the pheochromocytoma cell lines. The work which follows reports our findings on this catecholamine enzyme in PC12 and PCG2 cells. Considerable DBH activity does exist in these malignant cell lines but the enzyme behaves differently from that found in normal tissue. Initially, enzymatic activity does not increase linearly. Instead, activity is negligible at dilute protein concentrations. As protein concentration increases, activation of the enzyme occurs, and enzymatic activity increases linearly with further increases in protein concentration. A plateau in enzymatic activity is then observed at higher protein concentrations. In contrast to the normal medullary enzyme, DBH in neoplastic tissue exhibits negligible copper dependency. Finally, when DBH activity is maximized in PC12 cells, it becomes approximately equivalent to that observed in normal adrenal tissue but remains 45-fold greater than in PCG2 cells. MATERIALS AND METHODS
Tissue Male Sprague-Dawley rats (Simonsen, Gilroy, CA), 150 g, were killed by cervical dislocation. Adrenal glands were excised. Medullary tissue was isolated by nicking the cortical capsule and gently squeezing out the medulla. Tissue was frozen at -85 °C until used. The rat pheochromocytoma cell lines (courtesy of Dr. Eric Shooter, Department of Neurobiology) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 5% horse serum at 37 °C and 12% CO2/88% 02. Cells were serially passaged as needed, grown to confluency, and collected by low speed centrifugation. Cell pellets were frozen a t - 2 0 °C until used.
natant was diluted to a final volume of 5 ml using the s a m e buffer, and 200 fll was assayed for dopamine flhydroxylase activity as previously described is. Pheochromocytoma cells (4 x 107) were sonicated 15 s in 1 ml of the above buffer. Cellular debris was removed by centrifugation as described, and supernatants were diluted to a final volume of 2 ml. For enzyme linearity studies, varying amounts of normal and malignant extracts were diluted to 200/~l with 5 mM Tris-HCl, pH 7.4, and dopamine fl-hydroxylase activity was measured as above. To monitor the effects of copper on enzymatic activity, 10 ~l of copper at concentrations varying from 0.1 to 0.6 mM were added to 200 ~l of enzyme extract prior to assay. Final copper concentrations ranged from 3.2 to 19.4/~M. Protein concentration Protein was measured in norm~tl and neoplastic tissue extracts according to the procedure of Lowry et al. 10. Bovine serum albumin was used as a protein standard. RESULTS Dopamine fl-hydroxylase activity as a function of extract concentration Fig. 1 shows the linear increase in dopamine fl-hy-
t-
20,0
E C
~- 12.o ~,~
Enzymatic assay A pair of normal adrenal medulla was sonicated for 15 s in 5 vols. of 5 mM Tris-HCl, pH 7.4 containing 0.2% bovine serum albumin and 0.2% Triton X-100 (Sigma, St. Louis, MO) using a Heat Systems Ultrasonics cell disruptor at a setting of 4 (Heat Systems-Ultrasonics, Plainview, NY). Cellular debris was removed by centrifugation of the extracts for 2.5 min at 10,000 g in a Beckman microfuge, Model B (Beckman Instruments, Palo Alto, CA). The super-
16.o
8.0
< -r II1 4.o Q 0,2
0.4
0.6
08
1.0
1.2
[PROTEIN], pg/pl Fig. 1. Dopamine fl-hydroxylase activity in normal rat adrenals. Rat adrenal medulla was prepared and assayed for dopamine fl-hydroxylase as described in Materials and Methods. Changes in enzymatic activity were measured as a function of
increasing protein concentration.
146 droxylase activity with increasing concentrations of protein for normal rat adrenal medulla. Non-linear responses in enzymatic activity only occur when the concentration of enzyme is so high that it is no longer saturated with substrate (data not shown). By either diluting the extract or increasing the concentration of substrate, linearity can be restored. In contrast, Fig. 2 shows the non-linear increase in D B H activity in the PC12 and PCG2 cell lines, even at saturating levels of substrate. When enzymatic activity is compared in the p h e o c h r o m o c y t o m a cell lines to that in normal medullary tissue, a complex response is observed. At protein concentrations ranging from 0.22/~g/gl to 1.23 gg/pl, the neoplastic tissues are devoid of enzymatic activity while normal medullary tissue shows considerable D B H activity. At protein concentrations > 1.23 pg/pl, D B H is activated in neoplastic tissue, possibly as a result of enzyme cooperativity. The plateau in enzymatic activity observed for neoplastic tissue at protein concentrations > 4.00 pg/pl may represent maximum cooperativity or slightly subsaturating substrate conditions. Table I shows the m a x i m u m D B H activity observed for normal and neoplastic tissue and the protein concentrations at which this occurs. In neoplastic tissue, maximum enzyme activity necessitates approximately 4,fold higher protein concentrations. Under these conditions, D B H activity in PC12 cells is roughly equivalent to that in normal tissue and 45-fold greater than that in PCG2 cells. For comparison, the activity of another biogenic
to '~
PC12
Dopamine fl-hydroxylase activi(y in normal and neoplastic tissue at maximum activation Tissue was prepared as described in Methods. Protein was measured as described by Lowry et al. ]° and DBH activity was assayed as described by Wong et al. ts Tissue
Protein concentration (mg/ml)
D B H activity (nmol/mg protein~h)
Rat adrenal medulla PC12 cells PCG2 cells
1.02 4.76 4.03
65.00 + 0.86 76.01 + 3.44, 1.68 + 0. t0 b,c
p = 0.20, not significantly different from control. P = 0.01, significantly different from control. c p = 0.03, significantly different from PC12 cells. a
amine, PNMT, was measured in the extracts according to the procedure of Wong et al. 20. This enzyme shows a linear response with increasing protein concentrations similar to that observed for non-malignant tissue, even at very dilute protein concentrations. Fig. 3 illustrates this for PC12 cells. In PC12 cells, P N M T activity is about 1.5 times greater than in normal tissue (0.31 nmol/h/mg protein vs 0.18 nmol/h/mg protein). Copper dependency o f dopamine fl-hydroxylase in normal and malignant tissue Most tissues contain endogenous inhibitors for e:
"8 E e-
PCG2
0.3
eo.o
~__ 4 o o
I-L3 <
os
I-
o4
2o.o
i z,o
4.0
e.o
['PROTEIN],
0.4
I-->
~e
eoo
E >£ F--
TABLE I
2O
4 0
e.o
I,tg / ttl
Fig. 2. Dopamine fl-hydroxylase activity in PC12 and PCG2 cells. Extracts of PC12 and PCG2 cells were prepared and assayedfor dopamine 15-hydroxylase activity as described in Materials and Methods, Changes in e ~ a t i c activity were measured as a function of increasing protein concentration, 0 ~ DBH activity in the absence of copper; 0, DBH activity in the presence of copper.
0.2
0.1
z
n 1.0
2,0
I'PROTEIN
3.0
],
4.0
5.0
r i g / I.II
Fig. 3. PNMT activity in PC12 cells. Extracts of PC12 cells were prepared as described in Materials and Methods, and PNMT activity was measured as described by Wong et al. 2°. Changes in enzymatic activity were measured as a function of increasing protein concentration.
147
E
so
(n ::3
4o
>I.-
3o
> I~0 <
20
"1" t'n El
1o
I o5
I ,o
i 15
[Cu+Z],
I 20
i 25
I 30
r 6.0
I.IM
Fig. 4. Copper dependency of rat adrenal medullary dopamine fl-hydroxylase. Tissue extracts were prepared and assayed as described in Materials and Methods. 200-/A aliquots were assayed for enzymatic activity in the presence of copper. Final copper concentrations ranged from 3.2 to 19.4 ~M. Units = nmol/h.
specific requirement for copper is dependent on extract concentration. With more concentrated tissue preparations, higher concentrations of copper are necessary to optimize enzymatic activity as indicated by the rightward shift in the copper curve (Fig. 5). These results are in direct contrast to the copper requirements for both the PC12 and PCG2 cells (Fig. 6). Even though PC12 cells have much greater D B H activity than PCG2 cells, both cell lines show very little dependency on copper over a very broad range of copper concentrations (0.25-12.9 /~M and 1.84-12.9 pM, respectively). Furthermore, Fig. 2 shows that for the pheochromocytoma cell lines, D B H enzymatic activity is equivalent in the presence or absence of copper. DISCUSSION
D B H which block enzymatic activity. These inhibitors are inactivated by copper ion 7. Additionally, D B H activity itself is inhibited by copper when inhibitors are absent. Fig. 4 illustrates the copper dependency of dopamine fl-hydroxylase in normal rat adrenal medullary tissue. D B H activity plateaus at copper concentrations between 1 and 3/~M. At either extreme of this plateau region, enzymatic activity decreases markedly with changes in copper concentration. The
In the present study, we have examined d o p a m i n e fl-hydroxylase activity in PC12 and PCG2 cells, two clonal cell lines derived from the same rat pheochromocytoma 17. The reduced levels of D B H activity observed in these cell lines 6 may reflect tissue dilution rather than absolute amounts of enzyme, which, infact, may be quite substantial. At low protein concentrations, negligible enzyme activity is observed. Moreover, the addition of copper does not stimulate catalytic activity as in normal tissue. However, when more concentrated tissue extracts are used, activa-
2O PC12 o,
~o~
>0 PeG2
t.)
Q
.r,
a°
2.5
5.0
;'5
IO.O
12.5
[Cu+=]. MM Fig. 5. Dopamine fl-hydroxylase activity in normal rat adrenal medulla as a function of copper and extract concentrations. Tissue extracts were prepared and assayed as described in Materials and Methods. For each tissue concentration, a copper curve was run from 1.9 to 13.9/zM. l , 1-.7 dilution of extract; A, 1-.5 dilution of extract; Q, 1-.3 dilution of extract. Units = nmoi/h.
,!o
21o
3'or'!o,~0 [ C u + = ],
,I°
'0
3!0,~.!O ,'0
pM
Fig. 6. Copper dependency of dopamine/3-hydroxylase in the pheochromocytoma cell lines. Tissue extracts were prepared and dopamine fl-hydroxylase activity assayed as described in Materials and Methods. For the PC12 and PCG2 cells, copper curves were run from 0.25 to 12.9/zM and 1.84 to 12.9/~M, respectively. Units = nmol/h.
148 tion of the enzyme occurs, followed by a linear response of the enzyme with further increases in tissue concentration, and finally, a plateau in enzyme activity at high tissue concentrations. In the linear range, DBH increases approximately 60-fold in the PC12 cells and about 8-fold in the PCG2 cells. Maximum enzymatic activity in the PC12 cells is about 45-fold greater than in the PCG2 cells and roughly equivalent to normal adrenal medullary tissue. As in the case of dilute solutions, copper does not increase enzymatic activity in concentrated enzyme solutions either. Thus, endogenous inhibitors, present in normal tissue, may be absent in PC12 and PCG2 cells. Copper also does not directly inhibit DBH in these rat pheochromocytoma cell lines. In contrast, extracts of DBH from normal tissue show both a sharp copper optimum and inhibition of enzymatic activity by copper in the absence of enzyme inhibitors. That is, a critical concentration of copper ion eliminates the effects of endogenous inhibitors. Below this optimum, enzymatic activity is not maximal since insufficient copper is present to remove interfering inhibitors. Above the optimum, copper ion directly inhibits dopamine fl-hydroxylase catalytic activity. Thus, for normal tissue, copper titration curves are essential to the measurement of enzymatic activity. For rat neoplastic tissue, the presence or absence of copper is inconsequential. In contrast to rat neoplastic tissue, human pheochromocytomas show variable amounts of dopamine fl-hydroxylase depending on the tumor source. In some cases, minimal amounts of the enzyme are present1, 4,14 while in other cases, amounts of enzyme are equal to or greater than that found in normal medullary tissue 4,12. When DBH activity is correlated with dopamine (substrate) content or norepinephrine (product) content in human pheochromocytomas, little correspondence is observed 5. However, phenylethanolamine N-methyltransferase (PNMT) activity and epinephrine content (product) change proportionally. The lack of correspondence between D B H and its substrate and product provide indirect evidence for the presence of endogenous inhibitors of D B H in this species of neoplastic medullary tissue. In vitro measurements may reflect greater enzyme activity than actually exists in vivo since total activity is measured in vitro by inclusion of N-ethylmaleimide or copper
which inactivate endogenous enzyme inhibitors. Purification and characterization of DBH from a variety of normal and neoplastic tissue suggests that enzyme structure is conserved. Independent of source, the enzyme is a tetrameric glycoprotein consisting of four nearly identical subunits of molecular weight 71,000-78,000 daltons6,13,14.19. Two pairs of dimers are formed by covalent disulfide linkage of monomeric subunits. These dimers then associate through non-covalent bonding to form the native, tetrameric enzyme. However, there may be minor differences in interspecies molecular weight 11. Additionally, while antibodies generated against the rat and bovine enzymes cross-react with enzyme from both normal and malignant tissue in these two species, these same antibodies do not show equal avidity for the human enzyme from neoplastic adrenal medullary tissue. This would suggest that some heterology exists in the antigenic determinants of the enzymes isolated from the various sources. Although the interspecies differences in dopamine fl-hydroxylase are not extensive, the lack of copper dependency and absence of endogenous inhibitors for the enzyme in PC12 and PCG2 cells suggest that the regulation of D B H in these adrenal medullary malignant cell lines may be different from normal tissue and other neoplastic tissue. D B H is present at much lower concentrations than in the adrenal medulla. Hence, at dilute protein concentrations, one observes little enzymatic activity. As protein concentration is increased, activation of DBH occurs, followed by a plateau in enzymatic activity. This activation may arise as a consequence of positive cooperativity associated with subunit aggregation. That is, subunit association occurs as protein concentration increases. The proximity of the enzyme subunits may result in conformational changes which facilitate both dopamine binding and its conversion to norepinephrine. We have previously shown that dopamine fl-hydroxylase is regulated both by humoral and neuronal agents in normal rat adrenal medullary tissue 3. Glucocorticoids control D B H levels by regulating enzyme degradation while neuronal stimuli control DBH levels by regulating enzyme synthesis. The same appears to hold for two other cateeholamine biosynthetic enzymes, tyrosine hydroxylase and phenylethanolamine N-methyltransferase 2,15. In the
149 PC12 add P C G 2 cells, glucocorticoids affect catecholamine enzymes somewhat differently in that there does not appear to be concerted control of D B H , T H and PNMT. In the case of PC12 cells, dexamethasone stimulates tyrosine hydroxylase activity but not phenylethanolamine N-methyltransferase activity 9. In the case of PCG2 cells, this synthetic steroid potentiates the NGF-induction of tyrosine hydroxylase. In general, PCG2 cells more closely mimic the behavior of normal medullary tissue in vivo and in vitro. Further examination of potential regulatory agents and their effects on protein synthesis and degradation may help elucidate control mechanisms operant in these cell lines and distinguish how the regu-
latory mechanisms for dopamine fl-hydroxylase and other catecholamine enzymes in these malignant cells differ from one another, from normal tissue, and from other neoplastic tissue. ACKNOWLEDGEMENTS This work was supported by Grants M H 25998 and M H 00219 (R.S.D.A. to R . D . C . ) from the National Institute of Mental Health and grant PCM 80-11525 from the National Science Foundation. Many thanks to Ms. Terry Lehning for her excellent assistance in preparing this. manuscript.
REFERENCES 1 Cesselin, F., Pique, L., Bertagna, X., Benlot, C., Antreassian, J., Proeschel, M.F., Girard, F., Zogbi, F., Legrand, J.C. and Luton, J.P., Simultaneous evaluation of the catecholamine pathway and three opioid peptide-producing systems in human pheochromocytomas, Neuropeptides, 4 (1984) 175-182. 2 Ciaranelio, R.D., Regulation of phenylethanolamine N-methyltransferase, Biochem. Pharmacol., 27 (1978) 1895-1897. 3 Ciaraneilo, R.D., Wooten, G.F. and Axelrod, J., Regulation of dopamine fl-hydroxylase in rat adrenal glands, J. Biol. Chem., 250 (1975) 3204-3211. 4 Feldman, J.M., Phenylethanolamine N-methyltransferase activity determines the epinephrine concentration of pheochromocytomas, Res. Commun. Chem. Pathol. Pharmacol., 34 (1981) 389-398. 5 Feldman, J.M., Blaiock, J.A., Zern, R.T. and Wells, S.A., The relationship between enzyme activity and the catecholamine content and secretion of pheochromocytomas, J. Clin. Endocrinol. Metab., 49 (1979) 445-451. 6 Fong, J.C., Shenkman, L. and Goldstein, M., Purification of rat pheochromocytoma dopamine beta-hydroxylase, J. Neurochem., 34 (1980) 346-350. 7 Friedman, S. and Kaufman, S., 3,4-Dihydroxyphenylethylamine fl-hydroxylase, J. Biol. Chem., 240 (1965) 4763-4773. 8 Goodman, R. and Herschman, H.R., Nerve growth factormediated induction of tyrosine hydroxylase in a cional pheochromocytoma cell line, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 4587-4590. 9 Greene, L.A. and Tischler, A.S., Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 2424-2428. 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent,
J. Biol. Chem., 193 (1951) 265-275. 11 O'Connor, D.T., Frigon, R.P. and Sokoloff, R.L., Human catecholamine storage vesicle proteins, Clin. Exp. Hypertens, 4 (1982) 563-575. 12 Park, D.H., Kashimoto, T., Ebstein, R.P. and Goldstein, M., Purification and immunochemical characterization of dopamine beta-hydroxylase from human pheochromocytoma, Mol. Pharmacol., 12 (1976) 73-81. 13 Sabban, E.L., Greene, L.A. and Goldstein, M., Mechanism of biosynthesis of soluble and membrane-bound forms of dopamine beta-hydroxylase in PC12 pheochromocytoma cells, J. Biol. Chem., 258 (1983) 7812-7818. 14 Stone, R.A., Kirshner, N., Reynolds, J. and Vanamen, T.C., Purification and properties of dopamine beta-hydroxylase from human pheochromocytomas, Mol. Pharmacol., 10 (1974) 1009-1015. 15 Thoenen, H., Angeletti, P.U., Levi-Montalcini, R. and Kettler, R., Selective induction by nerve growth factor of tyrosine hydroxylase and dopamine fl-hydroxylase in the rat superior cervical ganglia, Proc. Natl. Acad. Sci. U.S.A., 68 (1971) 1598-1602. 16 Thoenen, H., Mueller, R.A. and Axelrod, J., Increased tyrosine hydroxylase activity after drug-induced alteration of sympathetic transmission, Nature (London), 221 (1969) 1264. 17 Warren, S. and Chute, R.N., Pheochromocytoma, Cancer, 29 (1972) 327-331. 18 Wong, D.L., Masover, S.J. and Ciaranelio, R.D., Regulation of dopamine fl-hydroxylase synthesis and degradation, J. Biol. Chem., 256 (1981) 758-764. 19 Wong, D.L., Speedie, M.K. and Ciaranello, R.D., Characterization of dopamine fl-hydroxylase subunit, Soc. Neurosci. Abstr., 10 (1984) 876. 20 Wong, D.L., Zager, E.L. and Ciaranello, R.D., S-Adenosylmethionine measurement by radioenzymatic assay and hormonal control of its regulatory properties in the CNS and periphery, J. Neurosci., 2 (1982) 758-764.
Brain Research, 358 (1985) 144-149 Elsevier
BRE 11185
Comparison of Dopamine fl-Hydroxylase Activity in Normal Rat Adrenal Medulla and Rat Pheochromocytoma Cells DONA L. WONG and ROLAND D. CIARANELLO
Laboratory of Developmental Neurochemistry, Departmentof Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.) (Accepted February 25th, 1985)
Key words: rat pheochromocytoma cell - - dopamine fl-hydroxylase activity
Dopamine fl-hydroxylase activity is reportedly negligible in malignant rat adrenal cell lines. However, in two pheochromocytoma cell lines, PC12 and PCG2, considerable amounts of this catecholamine enzyme exist but its characteristics differ from the enzyme found in normal rat tissue in two ways. First, in normal adrenal medullary tissue, dopamine fl-hydroxylase activity increases linearly with increasing protein concentration. Second, there is a stringent requirement for copper. Concentrations of copper above or below the optimum inhibit enzymatic activity. In contrast, in pheochromocytoma ceils, dopamine fl-hy&oxytase exhibits a sigmoidal response with increasing tissue content. At dilute protein concentrations where considerable dopamine fl-hydroxylase activity is observed in normal adrenal medullary extracts, enzymatic activity is negligible in the p h e o e h r o ~ o m a cell lines. As the protein concentration is increased, activation of the enzyme occurs, and enzymatic activity increases linearly with further increases in protein concentration. At very high concentrations of protein, enzymatic activity plateaus. For both PC12 and PCG2 cells, doparaine p-hydroxylase activity shows minimal copper dependency, suggesting that endogenous inhibitors present in normal tissue are absent in these malignant cell lines. Under conditions of maximum activation, the activity of the enzyme in PC12 ceils becomes equivalent to that in normal rat adrenal medulla but remains 45-fold greater than that in PCG2 cells.
INTRODUCTION
(PNMT) activity are elevated but no changes in C A T occur.
The pheochromocytoma clonal cell lines, PC129 and PCG28, were derived from a rat adrenal medullary tumor originally defined by Warren and Chute t7. Both cell lines hypersecrete catecholamines, but appear to be regulated differently. For example, cultured PC12 cells divide but do not form processes. In the presence of nerve growth factor (NGF), cell division ceases, and neurite outgrowth begins. Concomitantly, choline acetyltransferase (CAT) activity is induced. If the PC12 cells are simultaneously supplemented with glucocorticoids, the NGF-stimulated induction of C A T is blocked. However, tyrosine hydroxylase activity is stimulated, The latter is in contrast to the effect of glucocorticoids in normal chromaffin tissue where both tyrosine hydroxylase (TH) and phenylethanolamine N-methyltrausferase
Although derived from the same malignant tissue, cultured PCG2 cells behave oppositely 8. N G F does not induce the changes in morphology and growth rate in the P C G 2 cell line as observed in the PC12 cell line. Neurite outgrowth is not stimulated, but tyrosine hydroxylase induction occurs at levels comparable in magnitude to that seen in intact animals ~5. Dexamethasone potentiates this NGF-induced increase in TH in P C G 2 cells as it does in normal sympathetic neurons. In general, PCG2 cells appear to more closely mimic the response to growth factors and steroids seen in norma! sympathetic neurons, both inthe intact animal or grown in organ culture. Previous studies have suggested that dopmafine flhydroxylase ( D B H ) , the enzyme responsible for norepinephrine synthesis, is either absent or present in
Correspondence: D.L. Wong, Laboratory of Developmental Neurochemistry, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
145 negligible amounts in the pheochromocytoma cell lines. The work which follows reports our findings on this catecholamine enzyme in PC12 and PCG2 cells. Considerable DBH activity does exist in these malignant cell lines but the enzyme behaves differently from that found in normal tissue. Initially, enzymatic activity does not increase linearly. Instead, activity is negligible at dilute protein concentrations. As protein concentration increases, activation of the enzyme occurs, and enzymatic activity increases linearly with further increases in protein concentration. A plateau in enzymatic activity is then observed at higher protein concentrations. In contrast to the normal medullary enzyme, DBH in neoplastic tissue exhibits negligible copper dependency. Finally, when DBH activity is maximized in PC12 cells, it becomes approximately equivalent to that observed in normal adrenal tissue but remains 45-fold greater than in PCG2 cells. MATERIALS AND METHODS
Tissue Male Sprague-Dawley rats (Simonsen, Gilroy, CA), 150 g, were killed by cervical dislocation. Adrenal glands were excised. Medullary tissue was isolated by nicking the cortical capsule and gently squeezing out the medulla. Tissue was frozen at -85 °C until used. The rat pheochromocytoma cell lines (courtesy of Dr. Eric Shooter, Department of Neurobiology) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 5% horse serum at 37 °C and 12% CO2/88% 02. Cells were serially passaged as needed, grown to confluency, and collected by low speed centrifugation. Cell pellets were frozen a t - 2 0 °C until used.
natant was diluted to a final volume of 5 ml using the s a m e buffer, and 200 fll was assayed for dopamine flhydroxylase activity as previously described is. Pheochromocytoma cells (4 x 107) were sonicated 15 s in 1 ml of the above buffer. Cellular debris was removed by centrifugation as described, and supernatants were diluted to a final volume of 2 ml. For enzyme linearity studies, varying amounts of normal and malignant extracts were diluted to 200/~l with 5 mM Tris-HCl, pH 7.4, and dopamine fl-hydroxylase activity was measured as above. To monitor the effects of copper on enzymatic activity, 10 ~l of copper at concentrations varying from 0.1 to 0.6 mM were added to 200 ~l of enzyme extract prior to assay. Final copper concentrations ranged from 3.2 to 19.4/~M. Protein concentration Protein was measured in norm~tl and neoplastic tissue extracts according to the procedure of Lowry et al. 10. Bovine serum albumin was used as a protein standard. RESULTS Dopamine fl-hydroxylase activity as a function of extract concentration Fig. 1 shows the linear increase in dopamine fl-hy-
t-
20,0
E C
~- 12.o ~,~
Enzymatic assay A pair of normal adrenal medulla was sonicated for 15 s in 5 vols. of 5 mM Tris-HCl, pH 7.4 containing 0.2% bovine serum albumin and 0.2% Triton X-100 (Sigma, St. Louis, MO) using a Heat Systems Ultrasonics cell disruptor at a setting of 4 (Heat Systems-Ultrasonics, Plainview, NY). Cellular debris was removed by centrifugation of the extracts for 2.5 min at 10,000 g in a Beckman microfuge, Model B (Beckman Instruments, Palo Alto, CA). The super-
16.o
8.0
< -r II1 4.o Q 0,2
0.4
0.6
08
1.0
1.2
[PROTEIN], pg/pl Fig. 1. Dopamine fl-hydroxylase activity in normal rat adrenals. Rat adrenal medulla was prepared and assayed for dopamine fl-hydroxylase as described in Materials and Methods. Changes in enzymatic activity were measured as a function of
increasing protein concentration.
146 droxylase activity with increasing concentrations of protein for normal rat adrenal medulla. Non-linear responses in enzymatic activity only occur when the concentration of enzyme is so high that it is no longer saturated with substrate (data not shown). By either diluting the extract or increasing the concentration of substrate, linearity can be restored. In contrast, Fig. 2 shows the non-linear increase in D B H activity in the PC12 and PCG2 cell lines, even at saturating levels of substrate. When enzymatic activity is compared in the p h e o c h r o m o c y t o m a cell lines to that in normal medullary tissue, a complex response is observed. At protein concentrations ranging from 0.22/~g/gl to 1.23 gg/pl, the neoplastic tissues are devoid of enzymatic activity while normal medullary tissue shows considerable D B H activity. At protein concentrations > 1.23 pg/pl, D B H is activated in neoplastic tissue, possibly as a result of enzyme cooperativity. The plateau in enzymatic activity observed for neoplastic tissue at protein concentrations > 4.00 pg/pl may represent maximum cooperativity or slightly subsaturating substrate conditions. Table I shows the m a x i m u m D B H activity observed for normal and neoplastic tissue and the protein concentrations at which this occurs. In neoplastic tissue, maximum enzyme activity necessitates approximately 4,fold higher protein concentrations. Under these conditions, D B H activity in PC12 cells is roughly equivalent to that in normal tissue and 45-fold greater than that in PCG2 cells. For comparison, the activity of another biogenic
to '~
PC12
Dopamine fl-hydroxylase activi(y in normal and neoplastic tissue at maximum activation Tissue was prepared as described in Methods. Protein was measured as described by Lowry et al. ]° and DBH activity was assayed as described by Wong et al. ts Tissue
Protein concentration (mg/ml)
D B H activity (nmol/mg protein~h)
Rat adrenal medulla PC12 cells PCG2 cells
1.02 4.76 4.03
65.00 + 0.86 76.01 + 3.44, 1.68 + 0. t0 b,c
p = 0.20, not significantly different from control. P = 0.01, significantly different from control. c p = 0.03, significantly different from PC12 cells. a
amine, PNMT, was measured in the extracts according to the procedure of Wong et al. 20. This enzyme shows a linear response with increasing protein concentrations similar to that observed for non-malignant tissue, even at very dilute protein concentrations. Fig. 3 illustrates this for PC12 cells. In PC12 cells, P N M T activity is about 1.5 times greater than in normal tissue (0.31 nmol/h/mg protein vs 0.18 nmol/h/mg protein). Copper dependency o f dopamine fl-hydroxylase in normal and malignant tissue Most tissues contain endogenous inhibitors for e:
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os
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~e
eoo
E >£ F--
TABLE I
2O
4 0
e.o
I,tg / ttl
Fig. 2. Dopamine fl-hydroxylase activity in PC12 and PCG2 cells. Extracts of PC12 and PCG2 cells were prepared and assayedfor dopamine 15-hydroxylase activity as described in Materials and Methods, Changes in e ~ a t i c activity were measured as a function of increasing protein concentration, 0 ~ DBH activity in the absence of copper; 0, DBH activity in the presence of copper.
0.2
0.1
z
n 1.0
2,0
I'PROTEIN
3.0
],
4.0
5.0
r i g / I.II
Fig. 3. PNMT activity in PC12 cells. Extracts of PC12 cells were prepared as described in Materials and Methods, and PNMT activity was measured as described by Wong et al. 2°. Changes in enzymatic activity were measured as a function of increasing protein concentration.
147
E
so
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20
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1o
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i 15
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I 20
i 25
I 30
r 6.0
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Fig. 4. Copper dependency of rat adrenal medullary dopamine fl-hydroxylase. Tissue extracts were prepared and assayed as described in Materials and Methods. 200-/A aliquots were assayed for enzymatic activity in the presence of copper. Final copper concentrations ranged from 3.2 to 19.4 ~M. Units = nmol/h.
specific requirement for copper is dependent on extract concentration. With more concentrated tissue preparations, higher concentrations of copper are necessary to optimize enzymatic activity as indicated by the rightward shift in the copper curve (Fig. 5). These results are in direct contrast to the copper requirements for both the PC12 and PCG2 cells (Fig. 6). Even though PC12 cells have much greater D B H activity than PCG2 cells, both cell lines show very little dependency on copper over a very broad range of copper concentrations (0.25-12.9 /~M and 1.84-12.9 pM, respectively). Furthermore, Fig. 2 shows that for the pheochromocytoma cell lines, D B H enzymatic activity is equivalent in the presence or absence of copper. DISCUSSION
D B H which block enzymatic activity. These inhibitors are inactivated by copper ion 7. Additionally, D B H activity itself is inhibited by copper when inhibitors are absent. Fig. 4 illustrates the copper dependency of dopamine fl-hydroxylase in normal rat adrenal medullary tissue. D B H activity plateaus at copper concentrations between 1 and 3/~M. At either extreme of this plateau region, enzymatic activity decreases markedly with changes in copper concentration. The
In the present study, we have examined d o p a m i n e fl-hydroxylase activity in PC12 and PCG2 cells, two clonal cell lines derived from the same rat pheochromocytoma 17. The reduced levels of D B H activity observed in these cell lines 6 may reflect tissue dilution rather than absolute amounts of enzyme, which, infact, may be quite substantial. At low protein concentrations, negligible enzyme activity is observed. Moreover, the addition of copper does not stimulate catalytic activity as in normal tissue. However, when more concentrated tissue extracts are used, activa-
2O PC12 o,
~o~
>0 PeG2
t.)
Q
.r,
a°
2.5
5.0
;'5
IO.O
12.5
[Cu+=]. MM Fig. 5. Dopamine fl-hydroxylase activity in normal rat adrenal medulla as a function of copper and extract concentrations. Tissue extracts were prepared and assayed as described in Materials and Methods. For each tissue concentration, a copper curve was run from 1.9 to 13.9/zM. l , 1-.7 dilution of extract; A, 1-.5 dilution of extract; Q, 1-.3 dilution of extract. Units = nmoi/h.
,!o
21o
3'or'!o,~0 [ C u + = ],
,I°
'0
3!0,~.!O ,'0
pM
Fig. 6. Copper dependency of dopamine/3-hydroxylase in the pheochromocytoma cell lines. Tissue extracts were prepared and dopamine fl-hydroxylase activity assayed as described in Materials and Methods. For the PC12 and PCG2 cells, copper curves were run from 0.25 to 12.9/zM and 1.84 to 12.9/~M, respectively. Units = nmol/h.
148 tion of the enzyme occurs, followed by a linear response of the enzyme with further increases in tissue concentration, and finally, a plateau in enzyme activity at high tissue concentrations. In the linear range, DBH increases approximately 60-fold in the PC12 cells and about 8-fold in the PCG2 cells. Maximum enzymatic activity in the PC12 cells is about 45-fold greater than in the PCG2 cells and roughly equivalent to normal adrenal medullary tissue. As in the case of dilute solutions, copper does not increase enzymatic activity in concentrated enzyme solutions either. Thus, endogenous inhibitors, present in normal tissue, may be absent in PC12 and PCG2 cells. Copper also does not directly inhibit DBH in these rat pheochromocytoma cell lines. In contrast, extracts of DBH from normal tissue show both a sharp copper optimum and inhibition of enzymatic activity by copper in the absence of enzyme inhibitors. That is, a critical concentration of copper ion eliminates the effects of endogenous inhibitors. Below this optimum, enzymatic activity is not maximal since insufficient copper is present to remove interfering inhibitors. Above the optimum, copper ion directly inhibits dopamine fl-hydroxylase catalytic activity. Thus, for normal tissue, copper titration curves are essential to the measurement of enzymatic activity. For rat neoplastic tissue, the presence or absence of copper is inconsequential. In contrast to rat neoplastic tissue, human pheochromocytomas show variable amounts of dopamine fl-hydroxylase depending on the tumor source. In some cases, minimal amounts of the enzyme are present1, 4,14 while in other cases, amounts of enzyme are equal to or greater than that found in normal medullary tissue 4,12. When DBH activity is correlated with dopamine (substrate) content or norepinephrine (product) content in human pheochromocytomas, little correspondence is observed 5. However, phenylethanolamine N-methyltransferase (PNMT) activity and epinephrine content (product) change proportionally. The lack of correspondence between D B H and its substrate and product provide indirect evidence for the presence of endogenous inhibitors of D B H in this species of neoplastic medullary tissue. In vitro measurements may reflect greater enzyme activity than actually exists in vivo since total activity is measured in vitro by inclusion of N-ethylmaleimide or copper
which inactivate endogenous enzyme inhibitors. Purification and characterization of DBH from a variety of normal and neoplastic tissue suggests that enzyme structure is conserved. Independent of source, the enzyme is a tetrameric glycoprotein consisting of four nearly identical subunits of molecular weight 71,000-78,000 daltons6,13,14.19. Two pairs of dimers are formed by covalent disulfide linkage of monomeric subunits. These dimers then associate through non-covalent bonding to form the native, tetrameric enzyme. However, there may be minor differences in interspecies molecular weight 11. Additionally, while antibodies generated against the rat and bovine enzymes cross-react with enzyme from both normal and malignant tissue in these two species, these same antibodies do not show equal avidity for the human enzyme from neoplastic adrenal medullary tissue. This would suggest that some heterology exists in the antigenic determinants of the enzymes isolated from the various sources. Although the interspecies differences in dopamine fl-hydroxylase are not extensive, the lack of copper dependency and absence of endogenous inhibitors for the enzyme in PC12 and PCG2 cells suggest that the regulation of D B H in these adrenal medullary malignant cell lines may be different from normal tissue and other neoplastic tissue. D B H is present at much lower concentrations than in the adrenal medulla. Hence, at dilute protein concentrations, one observes little enzymatic activity. As protein concentration is increased, activation of DBH occurs, followed by a plateau in enzymatic activity. This activation may arise as a consequence of positive cooperativity associated with subunit aggregation. That is, subunit association occurs as protein concentration increases. The proximity of the enzyme subunits may result in conformational changes which facilitate both dopamine binding and its conversion to norepinephrine. We have previously shown that dopamine fl-hydroxylase is regulated both by humoral and neuronal agents in normal rat adrenal medullary tissue 3. Glucocorticoids control D B H levels by regulating enzyme degradation while neuronal stimuli control DBH levels by regulating enzyme synthesis. The same appears to hold for two other cateeholamine biosynthetic enzymes, tyrosine hydroxylase and phenylethanolamine N-methyltransferase 2,15. In the
149 PC12 add P C G 2 cells, glucocorticoids affect catecholamine enzymes somewhat differently in that there does not appear to be concerted control of D B H , T H and PNMT. In the case of PC12 cells, dexamethasone stimulates tyrosine hydroxylase activity but not phenylethanolamine N-methyltransferase activity 9. In the case of PCG2 cells, this synthetic steroid potentiates the NGF-induction of tyrosine hydroxylase. In general, PCG2 cells more closely mimic the behavior of normal medullary tissue in vivo and in vitro. Further examination of potential regulatory agents and their effects on protein synthesis and degradation may help elucidate control mechanisms operant in these cell lines and distinguish how the regu-
latory mechanisms for dopamine fl-hydroxylase and other catecholamine enzymes in these malignant cells differ from one another, from normal tissue, and from other neoplastic tissue. ACKNOWLEDGEMENTS This work was supported by Grants M H 25998 and M H 00219 (R.S.D.A. to R . D . C . ) from the National Institute of Mental Health and grant PCM 80-11525 from the National Science Foundation. Many thanks to Ms. Terry Lehning for her excellent assistance in preparing this. manuscript.
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