Neurochemical Research, VoL 16, No. 1, 1991, pp. 63-66
Stimulation of Carnitine Acetyltransferase in PC12 Cells by Nerve Growth Factor: Relationship to Choline Acetyltransferase Stimulation Helen L. W h i t e 1 and Philip W . Scates 1
(Accepted December 4, 1990)
The activity of carnitine acetyltransferase (acetyl-CoA:L-carnitine O-acetyllransferase) was found to be at least 50-fold higher than that of choline acetyltransferase in PC12 cells. Nerve growth factor stimulated both enzymes in a parallel manner with respect to concentration of NGF and culture time. The stimulation of both enzymes was completely inhibited by 10 ~M 6-thioguanine, an inhibitor of protein kinase N. Results are discussed with reference to the hypothesis that the two enzymes may be functionally related in neuronal cells. KEY WORDS: Acetyl-L-carnitine, 6-thioguanine, protein kinase N.
INTRODUCTION
stimulation of both enzymes in response to NGF was demonstrated.
PC12 cells are well-characterized rat pheochromocytoma cells that are converted to a neuronal phenotype when incubated with nerve growth factor (NGF) (1). This response to NGF, although not well understood, has been associated with neurite extension, regulation of protein kinases (2,3), and increases in certain enzyme activities (4-7), including choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6, CHAT), the enzyme which catalyzes synthesis of acetylcholine.
EXPERIMENTAL
PROCEDURE
Materials. PC12 cells (rat adrenal tumor) were from ATCC #CRL 1721. Culture media and additiveswere from Gibco Laboratories(Grand Island, NY); NGF (2.5-S) #100-700 from Boehringer Mannhein (Indianapolis, IN); antiserum to 2.5-S NGF (rabbit) from Collaborative Research, Inc. (Bedford, MA); acetyl-CoA [aeetyl-l-~4C] 48.8 Ci/ tool, and acetyl-CoA [acetyl-3H] 200 Ci/mol, from New England Nuclear (Wilmington, DE); L-[methyl-14C]carnitine (57 Ci/mol) from Amersham Corp. (Arlington Heights, IL); L-carnitine and 6-thioguanine from Sigma Chem. Co. (St. Louis, MO); choline from ICN Pharmaceuticals (Cleveland, OH); acetyl-L-carnitine from Life Science Resource Research (Milwaukee, WI); Whatman LK5D thin layer plates with preadsorbent origins from Whatman (Clifton, NJ); ion exchange resins from Biorad Corp. (Richmond, CA). PC12 Cell Culture Conditions. PC12 cells were cultured in DMEM supplemented with 10% fetal bovfne serum, 10% horse serum, 4 mM glutamine, 200 units/ml penicillin, 200 ~g/ml streptomycin, and 0.25 mM MEM non-essential amino acids (Gibco #320-1140AG). Cultures were split 1:4 every 5 days. For each experiment, exponentially dividing cells were plated in fresh medium on 25 mm diameter collagencoated dishes. After allowing one day for cell attachment, medium was replaced with serum-free medium, with or without NGF. Cells
In the present study the activities of ChAT and a related enzyme, carnitine acetyltransferase (acetyl-CoA:Lcarnitine O-acetyltransferase, EC 2.3.1.7, CarAT) were compared in PC12 cells. The enzymatic reaction catalyzed by CarAT may regulate the availability of acetylCoA in the cytoplasm, where it can serve as a substrate for acetylcholine synthesis by ChAT (8-11). It is, therefore, of interest that CarAT activity was much higher than ChAT activity in PC12 cells and that a parallel i Div. of Pharmacology, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709. 63
0364-3190/91/0100-0063506.50/0 9 1991 Plenum Publishing Corporation
White and Scares
64 were periodically examined for morphological changes, using an Olympus IMT-2 inverted research microscope. At specified times (672 hr at 37~ cells and media were transferred to 1.5 ml Eppendorf tubes. A 20 ~xlaliquot was removedfor cell countingand determination of viability by trypan blue exclusion. The remainder was pelleted in a microfuge. Cells were washed once with serum-free medium and finally resuspended in 30 tzl of distilled water containing 0.2 mM eserine. Enzyme Assays. Resuspended cells were lysed by 3 freeze-thaw cycles and 2 sec sonication on power setting 3, using a Heat Systems Ultronic Model W385 with cup horn attachment (Farmingdale, NY). ChAT was assayed at pH 7.0 in a total volume of 60 p.1 containing 20 ~1 of lysed cell suspension and final concentrations (mM) of potassiumn phosphate (10), EDTA (0.02), NaC1 (200) eserine (0.12), choline (0.5), and [a4C]acetyl-eoA(0.040; 0.2 ixei). Naphthylvinylpyridine (0.25 raM) was included in blank assays to specificallyinhibit CHAT, but not CarAT (12). Following a 20 min incubation at 37~ assay mixtures were applied to 0.5 x 3 cm columns of Bio-Rad AG1X8 resin, and the product, acetylcholine,was eluted directly into scintillation vials with 1.5 ml distiIled water, using a procedure described earlier (13). CarAT was assayed as above, with the following modifications: 10 ixl of lysate was used; L-carnitine (0.5 raM) was added instead of choline, naphthylvinylpyridine(0.25 raM) was included in all assays in order to block ChAT activity; and incubations were for 10 min at 37~ The labeled product of CarAT, acetyl-L-carnitine, was verified by thin-layerchromatography.Eluants from the AG1-X8 columns were dried under nitrogen and reconstituted in 50 txl water containing 20 ~g each of unlabeled standards, acetyl-L-carnitine and L-carnitine. These were spotted on silica gel G plates (Whatman LK5D) and developed with CHC13/methanol/acetone/aceticacid/water (160/52/60/48/ 28.8; vols). After drying at room temperature, plates were exposed to iodine vapor for visualization of standards and/or examined autoradiographically. Rr values for L-carnitine and acetyl-L-carnitine in this system were 0.15 and 0.28, respectively. Appropriate areas were then scraped for scintillation counting, after addition of EcoLitescintillation cocktail (ICN Biomedicals, Inc., Irvine, CA). Recoveryof a4C-labeled product from the thin-layer plates was approximately 63%.
RESULTS
CarAT in PC12 cells. To demonstrate the presence of CarAT in PC12 cells, cell lysates were incubated with [3H]acetyl-CoA and [14C]L-carnitine in the presence of naphthylvinylpyridine. Eluants from ion exchange columns were dried under nitrogen to reduce volumes, resuspended in the presence of unlabeled L-carnitine and acetyl-L-carnitine, and chromatographed on silica gel G thin-layer plates as described above. Regions of the plates corresponding with the internal standards were counted using a double-label program. As seen in Table I, the carnitine region contained mostly [14C]label, while both [3H] and [14C] were found in the acetylcarnitine region in an approximately equimolar ratio, as would be expected for the dual-labeled product. This peak was not observed when carnitine was omitted from the assays. Effect of NGF on ChAT and CarAT. The stim-
Table I. Formation of Acetyl-L-Carnitinefrom ?4C]L-Carnitine and [3H]Acetyl-CoAin PC12 Cell Lysates i
nmol Recovered Region of TLC Plate
[aH]
[14C]
Rf = 0.28 (Acetylcarnitine) Rf = 0.15 (Carnitine)
0.88
1.12
0.05
11.3
Lysates of PC12 cells were incubated for 20 min at 37~ with 25 nmol [t4C]L-carnitine and 2 nmol of [3H]acetyl-CoAprior to ion exchange separation and thin-layer chromatography. Regions of plates corresponding with location of internal standards were scraped and counted by scintillation techniques. Data are expressed as nmol of label recovered from indicated regions of thin-layer plates. Similar results were obtained in replicate experiments.
ulations of both ChAT and CarAT by increasing concentrations of NGF after 72 hr in culture are shown in Figure 1. ChAT and CarAT assays were performed on aliquots of the same cell lysates. Although the basal activity of CarAt in these cells was at least 50-fold greater than ChAT activity at the conditions employed, the stimulation by NGF showed a similar pattern for the two enzymes. This is also demonstrated in Figure 2 where the effect of 5 ng/ml NGF was followed as a function of time. The activities of both ChAT and CarAT were found to be different from control values after 6 hr incubation with NGF, and the percent stimulation increased with time thereafter. Neurite outgrowth in these cells was observed by microscopic evaluation at 24 hr with 5 ng/ml NGF and also increased with incubation time and with higher NGF concentrations. When the serine kinase inhibitor, 6-thioguanine, was included during incubations with PC12 cells, both basal and NGF,stimulated activities were inhibited. As seen in Table II, 6-thioguanine at 10 IxM caused at least 95% inhibition of ChAT and at least 50% inhibition of CarAT. The inhibition was dose-dependent (24-33% inhibition at 1 txM 6-thioguanine). NGF-stimulated neurite outgrowth was clearly inhibited at 3-10 IxM 6-thioguanine.
DISCUSSION The specific activity of ChAT in PC12 cells, based on mg protein, has been shown to increase with cell density and also with certain culture conditions (4-7). In the present study, PC12 cells were found to be more responsive to NGF when incubated at low serum concentrations. Therefore, all experiments concerning the effects of NGF were performed using serum-free me-
Stimulation of Carnitine Acetyltransferase in PC12 Cells by N G F
200
I
65
250
m
150
Z [-,
Stimulation of Carnitine Acetyltransferase in PC12 Cells by Nerve Growth Factor: Relationship to Choline Acetyltransferase Stimulation Helen L. W h i t e 1 and Philip W . Scates 1
(Accepted December 4, 1990)
The activity of carnitine acetyltransferase (acetyl-CoA:L-carnitine O-acetyllransferase) was found to be at least 50-fold higher than that of choline acetyltransferase in PC12 cells. Nerve growth factor stimulated both enzymes in a parallel manner with respect to concentration of NGF and culture time. The stimulation of both enzymes was completely inhibited by 10 ~M 6-thioguanine, an inhibitor of protein kinase N. Results are discussed with reference to the hypothesis that the two enzymes may be functionally related in neuronal cells. KEY WORDS: Acetyl-L-carnitine, 6-thioguanine, protein kinase N.
INTRODUCTION
stimulation of both enzymes in response to NGF was demonstrated.
PC12 cells are well-characterized rat pheochromocytoma cells that are converted to a neuronal phenotype when incubated with nerve growth factor (NGF) (1). This response to NGF, although not well understood, has been associated with neurite extension, regulation of protein kinases (2,3), and increases in certain enzyme activities (4-7), including choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6, CHAT), the enzyme which catalyzes synthesis of acetylcholine.
EXPERIMENTAL
PROCEDURE
Materials. PC12 cells (rat adrenal tumor) were from ATCC #CRL 1721. Culture media and additiveswere from Gibco Laboratories(Grand Island, NY); NGF (2.5-S) #100-700 from Boehringer Mannhein (Indianapolis, IN); antiserum to 2.5-S NGF (rabbit) from Collaborative Research, Inc. (Bedford, MA); acetyl-CoA [aeetyl-l-~4C] 48.8 Ci/ tool, and acetyl-CoA [acetyl-3H] 200 Ci/mol, from New England Nuclear (Wilmington, DE); L-[methyl-14C]carnitine (57 Ci/mol) from Amersham Corp. (Arlington Heights, IL); L-carnitine and 6-thioguanine from Sigma Chem. Co. (St. Louis, MO); choline from ICN Pharmaceuticals (Cleveland, OH); acetyl-L-carnitine from Life Science Resource Research (Milwaukee, WI); Whatman LK5D thin layer plates with preadsorbent origins from Whatman (Clifton, NJ); ion exchange resins from Biorad Corp. (Richmond, CA). PC12 Cell Culture Conditions. PC12 cells were cultured in DMEM supplemented with 10% fetal bovfne serum, 10% horse serum, 4 mM glutamine, 200 units/ml penicillin, 200 ~g/ml streptomycin, and 0.25 mM MEM non-essential amino acids (Gibco #320-1140AG). Cultures were split 1:4 every 5 days. For each experiment, exponentially dividing cells were plated in fresh medium on 25 mm diameter collagencoated dishes. After allowing one day for cell attachment, medium was replaced with serum-free medium, with or without NGF. Cells
In the present study the activities of ChAT and a related enzyme, carnitine acetyltransferase (acetyl-CoA:Lcarnitine O-acetyltransferase, EC 2.3.1.7, CarAT) were compared in PC12 cells. The enzymatic reaction catalyzed by CarAT may regulate the availability of acetylCoA in the cytoplasm, where it can serve as a substrate for acetylcholine synthesis by ChAT (8-11). It is, therefore, of interest that CarAT activity was much higher than ChAT activity in PC12 cells and that a parallel i Div. of Pharmacology, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709. 63
0364-3190/91/0100-0063506.50/0 9 1991 Plenum Publishing Corporation
White and Scares
64 were periodically examined for morphological changes, using an Olympus IMT-2 inverted research microscope. At specified times (672 hr at 37~ cells and media were transferred to 1.5 ml Eppendorf tubes. A 20 ~xlaliquot was removedfor cell countingand determination of viability by trypan blue exclusion. The remainder was pelleted in a microfuge. Cells were washed once with serum-free medium and finally resuspended in 30 tzl of distilled water containing 0.2 mM eserine. Enzyme Assays. Resuspended cells were lysed by 3 freeze-thaw cycles and 2 sec sonication on power setting 3, using a Heat Systems Ultronic Model W385 with cup horn attachment (Farmingdale, NY). ChAT was assayed at pH 7.0 in a total volume of 60 p.1 containing 20 ~1 of lysed cell suspension and final concentrations (mM) of potassiumn phosphate (10), EDTA (0.02), NaC1 (200) eserine (0.12), choline (0.5), and [a4C]acetyl-eoA(0.040; 0.2 ixei). Naphthylvinylpyridine (0.25 raM) was included in blank assays to specificallyinhibit CHAT, but not CarAT (12). Following a 20 min incubation at 37~ assay mixtures were applied to 0.5 x 3 cm columns of Bio-Rad AG1X8 resin, and the product, acetylcholine,was eluted directly into scintillation vials with 1.5 ml distiIled water, using a procedure described earlier (13). CarAT was assayed as above, with the following modifications: 10 ixl of lysate was used; L-carnitine (0.5 raM) was added instead of choline, naphthylvinylpyridine(0.25 raM) was included in all assays in order to block ChAT activity; and incubations were for 10 min at 37~ The labeled product of CarAT, acetyl-L-carnitine, was verified by thin-layerchromatography.Eluants from the AG1-X8 columns were dried under nitrogen and reconstituted in 50 txl water containing 20 ~g each of unlabeled standards, acetyl-L-carnitine and L-carnitine. These were spotted on silica gel G plates (Whatman LK5D) and developed with CHC13/methanol/acetone/aceticacid/water (160/52/60/48/ 28.8; vols). After drying at room temperature, plates were exposed to iodine vapor for visualization of standards and/or examined autoradiographically. Rr values for L-carnitine and acetyl-L-carnitine in this system were 0.15 and 0.28, respectively. Appropriate areas were then scraped for scintillation counting, after addition of EcoLitescintillation cocktail (ICN Biomedicals, Inc., Irvine, CA). Recoveryof a4C-labeled product from the thin-layer plates was approximately 63%.
RESULTS
CarAT in PC12 cells. To demonstrate the presence of CarAT in PC12 cells, cell lysates were incubated with [3H]acetyl-CoA and [14C]L-carnitine in the presence of naphthylvinylpyridine. Eluants from ion exchange columns were dried under nitrogen to reduce volumes, resuspended in the presence of unlabeled L-carnitine and acetyl-L-carnitine, and chromatographed on silica gel G thin-layer plates as described above. Regions of the plates corresponding with the internal standards were counted using a double-label program. As seen in Table I, the carnitine region contained mostly [14C]label, while both [3H] and [14C] were found in the acetylcarnitine region in an approximately equimolar ratio, as would be expected for the dual-labeled product. This peak was not observed when carnitine was omitted from the assays. Effect of NGF on ChAT and CarAT. The stim-
Table I. Formation of Acetyl-L-Carnitinefrom ?4C]L-Carnitine and [3H]Acetyl-CoAin PC12 Cell Lysates i
nmol Recovered Region of TLC Plate
[aH]
[14C]
Rf = 0.28 (Acetylcarnitine) Rf = 0.15 (Carnitine)
0.88
1.12
0.05
11.3
Lysates of PC12 cells were incubated for 20 min at 37~ with 25 nmol [t4C]L-carnitine and 2 nmol of [3H]acetyl-CoAprior to ion exchange separation and thin-layer chromatography. Regions of plates corresponding with location of internal standards were scraped and counted by scintillation techniques. Data are expressed as nmol of label recovered from indicated regions of thin-layer plates. Similar results were obtained in replicate experiments.
ulations of both ChAT and CarAT by increasing concentrations of NGF after 72 hr in culture are shown in Figure 1. ChAT and CarAT assays were performed on aliquots of the same cell lysates. Although the basal activity of CarAt in these cells was at least 50-fold greater than ChAT activity at the conditions employed, the stimulation by NGF showed a similar pattern for the two enzymes. This is also demonstrated in Figure 2 where the effect of 5 ng/ml NGF was followed as a function of time. The activities of both ChAT and CarAT were found to be different from control values after 6 hr incubation with NGF, and the percent stimulation increased with time thereafter. Neurite outgrowth in these cells was observed by microscopic evaluation at 24 hr with 5 ng/ml NGF and also increased with incubation time and with higher NGF concentrations. When the serine kinase inhibitor, 6-thioguanine, was included during incubations with PC12 cells, both basal and NGF,stimulated activities were inhibited. As seen in Table II, 6-thioguanine at 10 IxM caused at least 95% inhibition of ChAT and at least 50% inhibition of CarAT. The inhibition was dose-dependent (24-33% inhibition at 1 txM 6-thioguanine). NGF-stimulated neurite outgrowth was clearly inhibited at 3-10 IxM 6-thioguanine.
DISCUSSION The specific activity of ChAT in PC12 cells, based on mg protein, has been shown to increase with cell density and also with certain culture conditions (4-7). In the present study, PC12 cells were found to be more responsive to NGF when incubated at low serum concentrations. Therefore, all experiments concerning the effects of NGF were performed using serum-free me-
Stimulation of Carnitine Acetyltransferase in PC12 Cells by N G F
200
I
65
250
m
150
Z [-,
