Expression of Mitochondrial Uncoupling Proteins and GABA Signaling Molecules in Unstimulated and Nerve Growth Factor–Stimulated PC12 Cells: Models for Chromaffin Cells and Sympathetic Neurons

Abstract

PC12 cells are a cell line originating from rat adrenal medullary chromaffin (AMC) cells. They extend a neurite-like structure in response to nerve growth factor (NGF). Thus, unstimulated and NGF-stimulated PC12 cells are used as models for AMC cells and sympathetic ganglion cells, respectively. However, how closely unstimulated and stimulated PC12 cells resemble AMC cells and sympathetic neurons, respectively, has not been elucidated sufficiently. We explored these issues by using biochemical and immunocytochemical methods. AMC cells and PC12 cells selectively expressed uncoupling protein 3 (UCP3) and uncoupling protein 4 (UCP4), respectively, and glucocorticoid activity inhibited UCP4 expression in PC12 cells. PC12 cells expressed extremely low levels of chromaffin granule–associated proteins, whereas the amount of synaptophysin, a synaptic vesicle–associated protein, was much higher than that in the adrenal medulla. Similar to AMC cells, the muscarinic receptor type 1 was located at the cell periphery in unstimulated PC12 cells, and its expression was markedly enhanced by NGF. Furthermore, NGF stimulation abolished the expression of GABA signaling molecules in PC12 cells. The results suggest that the properties of unstimulated PC12 cells are between those of AMC cells and sympathetic ganglion cells and GABA signaling is intrinsic to AMC cells.

Keywords

chromaffin cell chromogranin GAD67 PC12 synaptophysin UCP

Introduction

PC12 cells are an immortalized cell line that originated from a rat adrenal pheochromocytoma from male rat adrenal medullary chromaffin (AMC) cells.¹ This cell line has been used as a model for AMC cells to elucidate the molecular mechanisms for catecholamine secretion,² membrane excitability,³ and signal transduction for muscarinic receptor–mediated secretion.^4,5 Furthermore, PC12 cells extend neurites in response to nerve growth factor (NGF),⁶ as do AMC cells.⁷ Because of this transformation into neuron-like cells, NGF-stimulated cells have also been employed as a model for sympathetic ganglion cells.⁸ Although how unstimulated and NGF-stimulated PC12 cells resemble AMC cells and sympathetic neurons, respectively, has been a subject of investigation for more than 40 years, there are still unanswered questions.

In the present study, we addressed this issue by examining the following four points. First, we explored the expression levels of secretary vesicle–related proteins. AMC cells contain two kinds of secretary vesicles:^9,10 large dense core vesicles (LDCVs) or chromaffin granules where catecholamines are stored, and small synaptic-like microvesicles (SLMVs). Secretion in AMC cells occurs through LDCVs,¹¹ and the function of SLMVs in AMC cells is unknown.¹² On the contrary, secretion in sympathetic ganglion cells occurs mainly through synaptic vesicles.¹³ Second, we examined uncoupling protein (UCP) expression. UCPs are localized in the inner mitochondrial membrane and play an important role in cell metabolism.¹⁴ AMC cells secrete catecholamines in response to hypoxia during delivery,^15,16 thereby enhancing the survival of the newborns. This hypoxia-induced secretion is the result of hypoxia detection by AMC cells themselves;^17–19 mitochondria play the essential role for hypoxia sensing in AMC cells.^17,20,21 UCPs, which comprise five isoforms,²² are indirectly implicated in the production of reactive oxygen species,²³ ATP consumption,^20,24,25 and others.¹⁴ Third, we examined muscarinic receptor expression. Muscarinic receptors consistently mediate synaptic transmission through generation of slow excitatory postsynaptic potentials (EPSPs) in sympathetic ganglion cells from frogs²⁶ to mammals.²⁷ On the contrary, the roles of muscarinic receptors in AMC cells are variable, depending on the animal species.^28,29 In rat AMC cells, nicotinic receptors mainly mediate neuronal transmission from sympathetic preganglionic nerve fibers, although muscarinic receptor stimulation induces catecholamine secretion in one fourth of the cells.^4,30,31 Finally, we explored the proteins involved in GABA signaling. GABA is thought to function as an auto/paracrine agent in AMC cells.^32–34 On the contrary, sympathetic ganglion chief cells express GABA_A receptors (GABA_ARs),^35,36 but not GABA-synthesizing enzymes.^37,38 In this experiment, we evaluated whether unstimulated and NGF-stimulated PC12 cells exhibit the properties of AMC cells and sympathetic neurons, respectively, by analyzing the expression of proteins involved in these four functions. The comparison to AMC cells and sympathetic neurons was made based on the present and previous findings with such cell types. The results will be helpful for researchers who use PC12 cells as a model for AMC cells or sympathetic neurons and will deepen our understanding of AMC cells as endocrine cells.

Materials and Methods

Animals

Male Wistar rats and Hartley guinea pigs (each, 250–300 g) were obtained from Kyudo (Toshu, Japan). The animals were housed in standard cages with free access to food and water and kept under a 12-h photoperiod. All procedures for the care and treatment of animals were carried out according to the Japanese Act on the Welfare and Management of Animals and the Guidelines for the Proper Conduct of Animal Experiments issued by the Science Council of Japan. The experiments were approved by the Institutional Animal Care and Use Committee of the University of Occupational and Environmental Health (permit AE07-012). All efforts were made to minimize the potential animal distress.

Immunocytochemistry

Acutely dissociated adrenal cells were obtained as described elsewhere.³⁹ Briefly, 10 rats and 10 guinea pigs were killed by cervical dislocation, and the adrenal glands were excised, and immediately immersed in ice-cold Ca²⁺-deficient balanced salt solution, in which 1.8 mM CaCl₂ was omitted from standard saline. The standard saline contained 137 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.5 mM MgCl₂, 0.53 mM NaH₂PO₄, 5 mM d-glucose, and 5 mM HEPES, and the pH of the solution was adjusted to 7.4 with 4 mM NaOH. The adrenal cortex was removed from the adrenal medulla with microscissors and forceps under stereoscopic observations, and the adrenal medulla was cut into two. The adrenal medullary preparations were then incubated in a 0.5% collagenase-containing Ca²⁺-free solution at 36°C for 15 min; the treatment was repeated twice in fresh enzyme solution. After the digestion, one or two pieces of preparation were placed in a dish with non-fluorescent glass (P35GC-0-14-C; MatTek, Ashland, MA), and cells were dissociated using fine needles under a microscope. Dissociated cells were allowed to attach to the glass for 30 min, fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.2) for 1 h, and pre-incubated in PBS with 5% fetal bovine serum (FBS) (172012; Sigma-Aldrich, Tokyo, Japan) and 0.3% Triton X-100 for 30 min. For indirect immunofluorescence evaluation, cells were treated overnight with rabbit anti-UCP2 antibody (Ab) (dilution, 1:200) (AB3040; Chemicon, Temecula, CA) (RRID: AB_2213932), rabbit anti-UCP3 Ab (1:200) (Affinity Bioreagents, Golden, CO), rabbit anti-UCP4 Ab (1:200) (sc-20815; Santa Cruz Biotechnology, Dallas, TX) (RRID: AB_2270488), or mouse anti-UCP4 Ab (1:200) (sc-365295; Santa Cruz Biotechnology) (RRID: AB_10843244).⁴⁰ After incubation, cells were washed three times with PBS and treated with the appropriate secondary Ab conjugated to Alexa Fluor 488 (1:200) (A-11029 and A-11034 for mouse and rabbit immunoglobulin G (IgG), respectively: Molecular Probes, Eugene, OR) (RRID: AB_2534088 and AB_2576217, respectively) or 546 (1:200) (A-11035 for rabbit IgG: Molecular Probes) (RRID: AB_2534093). Fluorescence was observed under a laser confocal microscope (LSM5 Pascal; Carl Zeiss, Tokyo, Japan). The objective oil-immersion lens had a magnification of ×63 and a numerical aperture of 1.4. For Alexa Fluor 488, a 514-nm laser was used, and the emission at 530–600 nm was observed (FITC-like fluorescence), whereas for Alexa Fluor 546, a 543-nm laser was used, and the emission above 560 nm was observed (rhodamine-like fluorescence). Fluorescence intensity per pixel was measured and expressed in arbitrary unit (au). To assess the expression level of proteins, such as UCPs, the averaged intensity of fluorescence in the nucleus, which was regarded as a background level because of nonspecific immunoreaction, was subtracted from fluorescence intensity at the cell periphery and in the cytoplasm, and the resulting intensity was averaged.

Immunoblotting

Five rats and five guinea pigs were killed by cervical dislocation, and the adrenal glands and brains were excised and immediately placed in ice-cold Ca²⁺-deficient balanced salt solution. The isolated adrenal medullae and brains were individually minced and homogenized with a Potter–Elvehjem homogenizer in 10 volumes of a solution containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and a protease inhibitor cocktail (set 1; Calbiochem, San Diego, CA). Homogenates were centrifuged at 500 × g for 10 min at 4°C to remove the nuclei, then the postnuclear supernatants were mixed with equal volumes of a buffer containing 25 mM Tris-HCl (pH 6.8), 4% SDS, and 20% glycerol. Protein concentrations in samples were determined using a BSA protein assay kit (Pierce, Rockford, IL). After the addition of 2-mercaptoethanol (final concentration, 5% v/v) and bromophenol blue (0.05% w/v) to the samples, proteins (about 5 µg) were separated by 10% (w/v) SDS-PAGE, and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% (w/v) nonfat powdered milk dissolved in PBS-T solution, which contained 2 mM NaH₂PO₄, 145 mM NaCl, and 0.1% Tween 20. The PVDF membrane was incubated overnight with goat anti-chromogranin A (CgA) Ab (sc-13090; Santa Cruz Biotechnology) (RRID: AB_2080982), rabbit anti-synaptotagmin 7 Ab,^41,42 mouse anti-synaptophysin Ab (SY 38; Progen, Heidelberg, Germany) (RRID: AB_1543003), rabbit anti-dopamine β-hydroxylase (DβH) Ab (AB1538; Chemicon) (RRID: AB_90751), rabbit vesicular GABA transporter (VGAT) Ab (VGAT11-s; Alpha Diagnostic, San Antonio, TX) (RRID: AB_2190109),¹² mouse anti-gephyrin Ab (sc-25311; Santa Cruz Biotechnology) (RRID: AB_627670),⁴³ mouse anti-actin Ab (MAB1501R; Sigma-Aldrich) (RRID: AB_2223041), mouse anti-glutamic acid decarboxylase (GAD) 65/67 Ab (73-508; NeuroMab, Davis, CA) (RRID: AB_2756510), or rabbit anti-UCP3 Ab (Affinity Bioreagents). The immunoreaction was detected by incubating the membrane with an appropriate secondary Ab linked to horseradish peroxidase (NA931 and NA934 for mouse and rabbit IgG, respectively: Global life Science Technologies Japan, Tokyo, Japan; A50-201P for goat IgG: Bethy laboratories, Montgomery, TX) (RRID: AB_772210, AB_772206, and AB_66756, respectively), and then with ECL-Plus Western Blotting Substrate (32132: ThermoFisher Scientific, Tokyo, Japan).

Cell Culture

PC12 cells¹² (RRID: CVCL_0481) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Life Technologies, Tokyo, Japan) supplemented with 10% FBS. INS-1 cells (RRID: CVCL_0352), a cell line originating from rat insulin-secreting cells,⁴⁴ were cultured in RPM1 1640 (Gibco) supplemented with 10% FBS, 10 mM HEPES, 11.1% glucose, 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol. For immunostaining, the cells were placed onto glass coverslips coated with collagen type I (BD Biosciences, San Jose, CA). The cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After washing three times with PBS, the cells were incubated in PBS containing 0.1% Triton X-100 for 30 min and then with PBS containing 1% FBS for 1 h at room temperature. The cells were treated overnight with rabbit anti-muscarinic type 1 receptor (M₁R) Ab (1:50) (Frontier Institute, Ishikari, Japan) (RRID: AB_2571791), mouse anti-M₂R Ab (1:50) (sc-71531; Santa Cruz Biotechnology) (RRID: AB_1126414), mouse anti-M₄R Ab (1:50) (MAB1576; Chemicon) (RRID: AB_2080217), or rabbit anti-M₅R Ab (1:100) (6391-2190; Biogenesis, Poole, UK), or rabbit anti-GAD65/67 Ab (1:100) (G5163: Sigma-Aldrich) (RRID: AB_477019) followed by the appropriate secondary Ab. We had previously confirmed the specificities of the anti-muscarinic receptor and anti-GAD65/67 Abs in our lab.^31,45 M₃R expression was not explored because of the lack of a specific Ab. For NGF stimulation, the cells were serum starved for 12 h before the application of 50 ng/ml NGF and cultured for 1 week in DMEM with (high K⁺ medium) or without the addition of 20 mM KCl.⁴⁶ To stimulate adenylate cyclase, PC12 cells were cultured in the presence of 50 µM forskolin with or without NGF for 3 days. Forskolin was dissolved in 0.1% dimethyl sulfoxide. For immunoblotting, the PC12 cells were cultured in 100-mm dishes, and the stimulated and unstimulated cells were washed with ice-cold PBS and lysed with ice-cold TNE/P buffer (10 mM Tris-HCl [pH 7.4], 1 mM EDTA, 150 mM NaCl, 1% nonidet P-40, 1 mM Na₃VO₄, and a protease inhibitor cocktail) and then subjected to centrifugation at 12,000 × g for 30 min at 4°C. The supernatant of the cell lysate was added to an equal volume of the SDS buffer.

RT-PCR

The expression of UCPs at the messenger RNA (mRNA) level was examined with RT-PCR, as described elsewhere.³¹ Briefly, the Micro-FastTrack Kit (Invitrogen, Life Technologies) was employed to isolate poly(A)⁺ RNA from the brains, adrenal medullae, and adrenal cortices of five rats and PC12 cells according to the manufacturer’s instructions. Oligo dT primer was utilized for the RT reaction to obtain complementary DNA (cDNA). Each 20-µl PCR solution contained 1.25 µl of DNA template, 4 pmol of primer, 2 mM of dNTPs, 0.5 units of rTaq (Takara, Otsu, Japan), and PCR buffer supplied with the kit. The following primers were used: 5′-GGAAAGGGACCTCTCCCAATGTT-3′ and 5′-AAGGAAGGCATGAACCCTTGTAGAA-3′ for Ucp2 (PCR product of 304 bp); 5′-CCTGCAGATCCAAGGGGAGAA-3′ and 5′-GGAGGGCATGAATCCTTTGTAGAAG-3′ for Ucp3 (PCR product of 706 bp); 5′-ACTGGAAGGGAAACCCTTGAGATT-3′ and 5′-CCATTCTCAGCCAGGAGGGTAGAA-3′ for Ucp4 (PCR product of 416 bp); 5′-CAGGCTGGATACCCAATATTC-3′ and 5′-AGGTTGGTTCATTATTCGGC-3′ for Ucp4 nested (PCR product of 209 bp). The PCR protocol consisted of an initial 3 min denaturation at 94°C and then 30–40 cycles of 30 sec of denaturation at 94°C, 30 sec of annealing at 54°C to 60°C, and 30 sec of extension at 72°C. PCR products were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide. To check the specificity of PCR, PCR was performed with distilled water instead of experimental samples under the same conditions as the experimental samples, and this control PCR with each pair of primers did not result in production of any bands.

Sources of Reagents

Collagenase, NGF, and forskolin were obtained from Yakult (Kunitachi, Japan), Wako (Osaka, Japan), and Sigma-Aldrich (Tokyo, Japan), respectively; a rabbit anti-synaptotagmin 7 Ab was kindly provided by Dr. M. Fukuda (Tohoku University, Sendai, Japan).

Statistics

The data were expressed as the mean ± standard error of the mean (SEM). The data with a normal distribution (Shapiro–Wilk test) were assessed with an unpaired Student’s t-test or a Bonferroni t-test. Otherwise, the Mann–Whitney rank sum test or the Kruskal–Wallis analysis of variance on ranks test was used. A p value of <.05 was defined a statistically significant difference. Statistical analysis was performed with Sigma Plot v13.0 software (Systat Software, San Jose, CA).

Results

Secretion-Related Proteins

We used immunoblotting to explore the extent to which PC12 cells exhibit the properties of the original rat AMC cells. The expression level of 70-kDa CgA, a major protein constituting chromaffin granules,⁴⁷ was extremely low in PC12 cells compared with the rat adrenal medulla homogenates (Fig. 1A). In parallel to low CgA expression, the 40- and 43-kDa bands of synaptotagmin 7,^41,48 a major Ca²⁺ sensor in catecholamine-storing chromaffin granules,^42,49 were minimally expressed in PC12 cells, whereas they were conspicuous in the rat adrenal medulla homogenates (Fig. 1B). In contrast to these proteins, the expression of 38-kDa synaptophysin, a marker protein for synaptic vesicles or SLMVs,^10,50 was five times higher in PC12 cells than in the adrenal medulla homogenates (Fig. 1C). These results raise the possibility that a significant portion of the secretory activity in PC12 cells occurs through SLMVs rather than through LDCVs.¹¹ Consistent with this notion, 83-kDa DβH,⁵¹ an enzyme mediating biosynthesis of noradrenaline from dopamine, showed very low expression in PC12 cell homogenates compared with adrenal medulla homogenates (Fig. 1D). Last, we investigated the molecular form of synaptophysin. Consistent with a previous study,⁹ the apparent molecular weight of synaptophysin in the brain was 34 kDa, whereas that in the adrenal medulla homogenates and PC12 cells was 38 kDa because of glycosylation (Fig. 1E). When PC12 cells were stimulated with NGF for 1 week, 30% ± 3% (n=3) of the synaptophysin molecules existed as the 34-kDa form, suggesting that some NGF-stimulated PC12 cells differentiate into neurons.

Figure 1.

Immunoblotting for chromogranin A, synaptotagmin 7, synaptophysin, and dopamine β-hydroxylase of rat adrenal medulla and PC12 cell homogenates. (A, B, and C) Immunoblotting for chromogranin A (CgA), synaptotagmin 7 (Syt7), and synaptophysin (Syn) and actin, respectively. Left and right panels represent immunoblots and histograms of relative amounts of CgA, Syt7, and Syn bands. The amounts of the bands in PC samples were expressed as fractions of those in rat adrenal medulla (AM). The data represent means ± SEM of n=4, n=4, and n=3 for CgA, Syt7, and Syn, respectively. The same amounts of proteins (ca 5 µg) of rat AM and PC12 cells homogenates (PC) were loaded in gels and separated by electrophoresis and transferred to PDVF membranes. The PDVF membranes were treated overnight with primary Abs and then with secondary Abs. The immunoreaction was visualized with ECL-Plus. (D) Immunoblotting for dopamine β-hydroxylase (DβH) of rat AM and PC12 homogenates. (E) Immunoblotting for Syn and actin of rat brain (B), unstimulated (CON) and nerve growth factor–stimulated PC12 cells homogenates (NGF).

UCPs

UCPs are in the inner membrane of mitochondria and function as regulated proton channels or transporters; thus, they play an important role in energy metabolism.^14,52 There are five UCP isoforms, which exhibit selective expression in different tissues.²² Therefore, we explored UCP isoforms expressed in AMC and PC12 cells with immunocytochemistry (Fig. 2A). We quantitatively assessed the amount of UCP-like immunoreactive material in PC12 and rat AMC cells, which have common genomic information (Fig. 2B). Consistent with previous findings,⁵³ we immunocytochemically detected UCP2-like immunoreactive material in the cytoplasm in INS-1 cells derived from rat pancreatic β cells⁴⁴ (data not shown), indicating that the anti-UCP2 Ab used was immunoactive. This Ab did not generate immunofluorescence in the cytoplasm of guinea pig AMC (n=8), rat AMC (n=6), or PC12 (n=6) cells. In contrast to UCP2, we detected UCP3-like immunoreactive material in the cytoplasm of guinea pig (n=17) and rat AMC (n=14) cells, but not in PC12 cells (n=9). We examined the expression of UCP4 with two different anti-UCP4 Abs. The treatment with a rabbit anti-UCP4 Ab produced UCP4-like immunoreactive material in the cytoplasm in PC12 cells (n=3), but not in guinea pig (n=13) or rat AMC (n=11) cells. We reproduced these results by using a monoclonal anti-UCP4 Ab (data not shown), which has been reported to be specific for UCP4.⁴⁰

Figure 2.

Immunocytochemical and RT-PCR analyses for uncoupling protein expression in adrenal medullary chromaffin and PC12 cells. (A) Immunocytochemical staining of uncoupling protein (UCP) 2, UCP3, and UCP4 in PC12, guinea pig (GP), and rat adrenal medullary chromaffin (AMC) cells. The cells were treated with rabbit anti-UCP Abs and then with secondary Abs coupled with Alexa Fluor 488 or 546. Left and right in each panel represent confocal image of fluorescence and merge of fluorescence and differential interference contrast (DIC) images. Green and red indicate FITC- and rhodamine-like fluorescence, respectively. The bar in each panel represents 2 µm. (B) Summary of specific UCP-like immunofluorescence in PC12 and rat AMC cells. The intensity of specific UCP-like immunofluorescence per pixel in the cytoplasm was averaged and expressed in arbitrary unit (au). The data represent means ± SEM of PC12 (open) and rat AMC cells (closed) (n=6 and n=6 for UCP2; n=14 and n=19 for UCP3; n=3 and n=11 for UCP4, respectively). (C) Immunoblotting for UCP3 of the homogenates of guinea pig brain (B), adrenal cortex (AC), and adrenal medulla (AM). The same amounts of samples were loaded for each lane. (D) RT-PCR analysis for UCP expression in rat B, AC, and AM. Note that PCR products of 304, 706, 416, and 209 bp for Ucp2, Ucp3, Ucp4, and Ucp4 nested, respectively, were amplified in B, AC, AM, and/or PC12 preparations. M shows size markers with steps of 100 bp and intense bands represent 500 bp. Note that a PCR product was amplified only for Ucp3 in the AM sample, whereas that was only for Ucp4 in the PC12 sample.

We further performed immunoblotting with the anti-UCP3 Ab. As is evident in Fig. 2C, a 32-kDa band⁵³ of UCP3 was detected in each homogenate of guinea pig adrenal medulla, adrenal cortex, and brain; we noted the same result in the rat samples (data not shown). Next, we examined the expression of UCP isoforms at the mRNA level with RT-PCR. Figure 2D shows that PCR products for UCP2 were amplified in the rat adrenal cortex, but not in the brain, adrenal medulla, or PC12 samples, whereas those for UCP3 were recognized in the adrenal cortex, adrenal medulla, and brain, but not in the PC12 samples. As expected,²² UCP4 PCR products were amplified in the brain and PC12 samples, but not in the adrenal medulla or adrenal cortex samples. The results obtained with analyses on the protein and mRNA levels indicate that UCP3 and UCP4 are selectively expressed in AMC and PC12 cells, respectively.

High concentrations of glucocorticoids to which AMC cells are exposed in vivo play a crucial role for the function of AMC cells.⁵⁴ Thus, we investigated the effects of dexamethasone, a synthetic glucocorticoid, on the expression of UCP4 in PC12 cells. As is evident in Fig. 3, the amount of UCP4-like immunoreactive material in the cytoplasm diminished in a time-dependent manner in the presence of dexamethasone. When we cultured PC12 cells in medium containing 5 µM dexamethasone for 1 week,⁵⁵ the level of UCP4-like immunofluorescence in the cytoplasm significantly decreased by 45%.

Figure 3.

Inhibition of UCP4 expression in PC12 cells by dexamethasone. (A) Immunocytochemical staining of UCP4-like immunoreactive material in PC12 cells cultured in the absence (control) and presence of 5 µM dexamethasone for 3 days (+DM3d) and 7 days (+DM7d). The cells were treated with a rabbit anti-UCP4 Ab and then with a secondary Ab coupled with Alexa Fluor 488. Left and right in each panel represent confocal image of fluorescence and merge of fluorescence and DIC images. The bar in each panel represents 2 µm. (B) Summary of UCP4-like fluorescence in untreated (control) and dexamethasone-treated (+DM3d and +DM7d) PC12 cells. The intensity of specific UCP4-like immunoreactive material in the cytoplasm was averaged and expressed in arbitrary unit (au). The data represent means ± SEM of 10, 9, and 8 cells for Control, +DM3d, and +DM7d, respectively. ns stands for not significant.

Muscarinic Receptor Subtypes

Although muscarinic receptors mediate very little synaptic transmission from sympathetic preganglionic nerve fibers to rat AMC cells, muscarinic receptor stimulation does induce catecholamine secretion, and M₁R-, M₄R-, and M₅R-like immunoreactive material are present in rat AMC cells.^29,31 Moreover, M₁R is involved in generation of slow EPSPs in rat sympathetic ganglion cells.⁵⁶ Thus, we examined the expression of muscarinic receptor subtypes in PC12 cells by performing immunocytochemistry with isoform-specific Abs.³¹ NGF stimulation has been reported to decrease M₁R expression.⁵⁷ However, immunoblotting showed that the 64-kDa M₁R band⁵⁸ in NGF-simulated PC12 cells tended to be larger than that in unstimulated cells (Fig. 4A and B). This expression was further enhanced in high K⁺ medium (Fig. 4A and B), in which NGF-induced differentiation into sympathetic neurons is facilitated.^46,59 Immunocytochemistry revealed that the amount of M₁R-like immunoreactive material in stimulated cells was significantly higher than that in unstimulated cells, and part of M₁R-like immunofluorescence was located at the cell periphery in unstimulated and NGF-stimulated cells (Fig. 4C, D, F, and G). This peripheral distribution appeared to be enhanced in the presence of 20 mM K⁺ (Fig. 4E and F). Consistent with previous findings,⁵⁷ M₂R- and M₄R-like immunoreactive material increased with NGF stimulation and was preferentially located in a punctate-like manner at the cell periphery (Fig. 4C, D, F, and G). In contrast to M₁R, M₂R, and M₄R, the majority of M₅R-like immunoreactive material was present in the cytoplasm in a reticular manner (Fig. 4D), suggesting the presence of M₅R-like immunoreactive material in the endoplasmic reticulum.⁶⁰

Figure 4.

Immunoblotting and immunostaining for muscarinic receptor subtypes in unstimulated and NGF-stimulated PC12 cells. (A) Immunoblotting for muscarinic receptor type 1 (M₁R) and actin of the homogenates of unstimulated (CON) and NGF-stimulated PC12 cells with (NGF + K) or without high K⁺ (NGF). The same amounts of proteins were loaded for each lane. (B) Histogram of relative amounts of M₁R band. The amounts of M₁R band in NGF and CON preparations were expressed as fractions of that in NGF + K. The data represent means ± SEM of n=4. (C and D) Immunostaining for M₁R, M₂R, M₄R, and M₅R in unstimulated and NGF-stimulated PC12 cells, respectively. (E) Immunostaining for M₁R in NGF-stimulated PC12 cells in the presence of 20 mM K⁺. Left and right in each panel represent confocal image of fluorescence and merge of fluorescence and DIC images, respectively. The cells were treated with a muscarinic receptor subtype–specific Ab and then with an appropriate secondary Ab coupled with Alexa Fluor 488. The bar in each panel represents 5 µm. (F) Histogram of fractions of muscarinic receptor subtype-like immunoreactive material at the cell periphery. The fraction (%) was obtained by dividing the amount of specific immunoreactive material at the cell periphery over the total amount at the cell periphery and in the cytoplasm. The data represent means ± SEM of n=5, n=4, n=4, n=4, and n=4 for M₁ NGF, M₁ NGF +K, M₂ NGF, M₄ NGF, and M₅ NGF, respectively. (G) Histogram of the intensity of muscarinic receptor subtype-like immunofluorescence per pixel in unstimulated (open) and NGF-stimulated cells (closed). The data represent means ± SEM of unstimulated and NGF-stimulated cells (n=4 and n=5 for M₁R, n=4 and n=4 for M₂R, n=4 and n=4 for M₄R, and n=3 and n=4 for M₅R, respectively). The intensity of specific muscarinic receptor subtype-like fluorescence per pixel at the cell periphery and in the cytoplasm was averaged and expressed in arbitrary unit (au).

Proteins Involved in GABA Signaling

The proteins involved in GABA signaling are expressed in rat AMC cells, but not in sympathetic ganglion chief cells.^37,38 Thus, we examined GABA-related proteins in unstimulated and NGF-stimulated PC12 cells. In addition, we examined these proteins in PC12 cells in the presence of dexamethasone, which is thought to facilitate differentiation of the cells into the endocrine phenotype. Our previous experiment with RT-PCR⁴⁴ revealed that mRNA encoding GAD67, one of the GABA-synthesizing enzymes (GAD65 and GAD67),^61,62 is expressed in PC12 cells. Consistent with this result, we detected a 67-kDa GAD band in unstimulated PC12 cells (Fig. 5A and B). Intriguingly, this band was absent in the NGF- or dexamethasone-stimulated PC12 cells. Similarly, we detected 50-kDa VGAT,⁶³ a transporter involved in GABA uptake into secretory vesicles, in the unstimulated PC12 homogenates, but not in the NGF-stimulated PC12 (Fig. 5C and D). In contrast to GAD, the amount of VGAT was maintained in dexamethasone-stimulated PC12 cells. We detected gephyrin with an apparent molecular weight of 98 kDa⁶⁴—one of the proteins anchoring GABA_ARs to the subsynaptic membrane in the brain^65,66—in unstimulated PC12 cells. However, NGF stimulation abolished the expression of gephyrin in PC12 cells, and the expression level of gephyrin in PC12 cells was not maintained by dexamethasone stimulation.

Figure 5.

Immunoblot analyses of expression of proteins involved in GABA signaling in unstimulated, NGF-, and dexamethasone-stimulated PC12 cells. (A, C, and E) Immunoblotting for GAD67, vesicular GABA transporter (VGAT), gephyrin, and actin of unstimulated (CON), NGF-, and dexamethasone (DM)-stimulated PC12 cells. (B, D, and F) Histograms of relative amounts of GAD67, VGAT, and gephyrin bands. The data represent means ± SEM of n=6 (B), n=6 (D), and n=4 (F). Amounts of GAD67, VGAT, and gephyrin bands detected in NGF- and DM-stimulated PC12 cells homogenates were expressed as fractions of each band in unstimulated PC12 cells.

Immunoblotting suggested that GAD67 expression is almost abolished in NGF-induced differentiation of PC12 cells into neuron-like cells. Thus, this issue was further explored by immunocytochemistry. As reported previously,⁶⁰ GAD67-like immunoreactive material significantly increased in PC12 cells treated with forskolin, a adenylate cyclase activator, for 3 days (Fig. 6A, B, and E). On the contrary, treatment of PC12 cells with NGF for 1 week resulted in differentiation into sympathetic neuron-like cells with the extension of neurites and almost abolished the production of GAD67-like immunoreactive material (Fig. 6C and E). However, forskolin treatment even in the presence of NGF produced a marked increase in GAD67-like fluorescence (Fig. 6D and E). These results suggest that differentiation of PC12 cells into sympathetic ganglion cells in response to NGF results in a decrease in GAD67 expression.

Figure 6.

Immunocytochemical analysis of GAD67 expression in PC12 cells treated with or without NGF and/or forskolin. (A and B) Untreated (absence) and forskolin (Forsk)-treated PC12 cells, respectively. (C and D) NGF-stimulated PC12 cells in the absence and presence of Forsk (Forsk), respectively. Left, middle, and right columns represent confocal image of GAD67-like fluorescence, DIC image, and merge of fluorescence and DIC images, respectively. The cells were treated with a rabbit anti-Gad65/67 Ab and then with a secondary Ab coupled with Fluor Alexa 488. The bar in each panel represents 5 µm. (E) Summary of the intensity of GAD-like immunofluorescence in the cytoplasm of the cells shown in A, B, C, and D. The data represent means ± SEM of n=18, n=15, n=16, and n=15 for A, B, C, and D, respectively. The intensity of specific GAD-like fluorescence per pixel in the cytoplasm was averaged and expressed in arbitrary unit (au).

Discussion

NGF-Induced Differentiation

Survival of sympathetic ganglion cells depends on a survival signal initiated by NGF, which is taken up into the nerve terminal and transported to the cell body in a retrograde manner,⁶⁷ and NGF facilitates the extension of neurites in PC12 cells. These findings suggest that NGF induces PC12 cells to differentiate into sympathetic neuron–like cells. This notion is supported by the fact that AMC and sympathetic ganglion cells are developmentally close.⁶⁸ M₁R activation in sympathetic ganglion cells inhibits M-type K⁺ channels, with consequent depolarization,⁵⁶ whereas M₄R stimulation suppresses voltage-dependent Ca²⁺ channels through a membrane-delimited manner.^56,69 Consistent with these findings, NGF stimulation increased membrane expression of M₁R and M₄R in PC12 cells. Selective localization of M₁Rs at the cell periphery was conspicuously enhanced by NGF in the high K⁺ medium; a high K⁺ ion concentration is known to facilitate differentiation of PC12 cells into catecholaminergic neurons.⁷⁰ However, the enhanced expression of M₁R in response to NGF is opposite to a previous report where NGF stimulation upregulated M₂R and M₄R and downregulated M₁R in PC12 cells.⁵⁷ This contradiction may be due to a difference in the commercially available Abs used in the current and previous studies. The immunoreactivity of the anti-M₁R Ab used in the current study was confirmed in the present study (Fig. 4A) and in a previous report.³¹

It is worth noting that M₁R mediates slow EPSPs in sympathetic neurons of various animals,⁷¹ whereas the involvement of muscarinic receptors in the synaptic transmission in AMC cells depends on the animal species.^30,31,72 In rat AMC cells, muscarinic receptor–mediated transmission might occur under severe stressful conditions,^31,73 whereas such a restriction may not be the case for guinea pig AMC cells.⁷² The finding that M₁R expression in PC12 cells was enhanced under conditions where the differentiation into catecholaminergic cells is facilitated suggests that in the rat, M₁R-mediated synaptic transmission plays a more important role in sympathetic neurons than in AMC cells.

A Model of AMC Cell

The present experiment clearly revealed that the expression levels of the proteins involved in catecholamine synthesis, storage, and secretion were conspicuously low in PC12 cells compared with AMC cells. This outcome is not due to immaturity of PC12 cells as endocrine cells. The expression of synaptophysin, a marker of synaptic vesicles or SLMVs,^9,10 was five times higher in PC12 cells compared with AMC cells. However, the molecular form of synaptophysin in unstimulated PC12 cells was not the neuronal type,⁹ and NGF stimulation facilitated its expression of the neuronal form of synaptophysin in PC12 cells. The results suggest that unstimulated PC12 cells have properties between AMC cells and sympathetic ganglion cells. This notion is supported by the differential expression of UCP isoforms in AMC cells and PC12 cells.

The UCP family consists of five isoforms:²² UCP1 is mainly detected in brown adipose tissue (BAT) and is involved in thermogenesis; UCP2 is present in various types of cells, such as endocrine cells and lymphocytes;^22,74 UCP3 is expressed in skeletal muscles and adult cardiac myocytes and is involved in fatty acid transport in mitochondria.⁷⁵ There is a close relationship between the UCP3 expression level and the extent of fatty acid β-oxidation.⁷⁶ UCP4 and UCP5 are selectively expressed in neurons.^77,78 Immunocytochemistry with an anti-UCP3 Ab, whose specificity we confirmed (Fig. 2C), revealed UCP3-like immunoreactive material in AMC cells but not in PC12 cells. On the contrary, immunocytochemistry with two different anti-UCP4 Abs showed UCP4-like immunoreactive material in PC12 cells but not in AMC cells. The immunocytochemical findings are consistent with the RT-PCR analysis. These results indicate that UCP3 and UCP4 are selectively expressed in AMC cells and PC12 cells, respectively. It would be worth considering the physiological significance of UCP3 expression in AMC cells. This isoform is assumed to be involved in fatty acid transport in mitochondria,⁷⁶ and thus increase oxidative phosphorylation. Thus, UCP3 expression may help the cells to survive and function under glucopenic conditions, such as starving, because fatty acids supplied from the adipose tissue can be used as a fuel source.

UCPs are indirectly involved in a decrease in the cellular ATP content in response to hypoxia. Under hypoxic conditions, an increase in the mitochondrial H⁺ concentration due to H⁺ influx through UCPs is compensated by the reversed operation of F₁F₀-ATPase with ATP consumption.^20,24 There is evidence that a decrease in cellular ATP is somehow involved in the mechanism by which O₂-sensing cells detect hypoxia.^20,25,79,80 In the present study, UCP3 was expressed in adult rat and guinea pig AMC cells, but not in PC12 cells. These results suggest that there is no difference among the UCP isoforms regarding their role in hypoxia detection. First, a short exposure to hypoxia and mitochondrial inhibitors results in significant catecholamine secretion in adult guinea pig AMC cells,^19,25 but not in adult rat AMC cells.^18,25 Second, PC12 cells, which express UCP4, secrete catecholamines in response to hypoxia and mitochondrial inhibitors.^81,82

Intriguingly, the expression of UCP4, one of the neuronal isoforms,^77,78 in PC12 cells was suppressed by culturing them in the presence of dexamethasone, as was noted for the expression of the GABA_AR α1 subunit.⁴³ Glucocorticoids are known to suppress neurite extension of NGF-stimulated PC12 cells.⁸³ These results indicate that glucocorticoid activity is involved in the suppression of neuronal differentiation. Thus, exposure to high glucocorticoid concentrations is necessary for AMC cells to function as endocrine cells that secrete catecholamines.

Intermediate properties of PC12

The expression of proteins involved in GABA signaling in unstimulated PC12 cells provides further evidence for the intermediate properties of PC12 cells. Table 1 represents a summary of protein expression in AMC cells and unstimulated and NGF-stimulated PC12 cells, which was examined in the present and previous experiments.^12,33,43 Immunoblotting and immunocytochemistry revealed that GAD67, VGAT, and gephyrin were expressed in unstimulated PC12 cells, but not in NGF-stimulated cells. Our^12,33 and other’s research³² have shown that GABA produced by GAD67 is taken up into chromaffin granules via VGAT to function as an auto/paracrine factor in AMC cells. On the other hand, gephyrin is the major protein that mediates the anchoring of GABA_ARs to the subsynaptic membrane in brain neurons.^65,66 This scaffolding protein is not expressed in rat AMC cells.⁴³ Thus, it is not clear why gephyrin is expressed in unstimulated PC12 cells. However, it is worth noting that PC12 cells express the α3 and the α1 subunits of GABA_ARs.⁴³ α1 is the major α subunit of GABA_ARs mediating inhibitory postsynaptic potentials (IPSPs) in adult brain neurons,⁸⁴ whereas α3 is one of the α subunits constituting GABA_ARs involved in IPSPs during the neonatal period.⁸⁴ The α3 subunit in the GABAergic subsynaptic membrane in a certain brain area is replaced with the α1 subunit during brain development.^85–87 Culturing PC12 cells in the presence of dexamethasone decreased α1 expression and increased α3 expression.⁴³ Interestingly, VGAT expression was maintained in the presence of dexamethasone, but gephyrin expression was not. Because the α1 subunit has a binding site for gephyrin,⁸⁸ the results suggest that α1 expression is somehow coupled with gephyrin expression, with the consequent facilitation of inhibitory synaptic transmission. The finding that GAD67, VGAT, and gephyrin were not expressed in NGF-stimulated PC12 cells further suggests that NGF differentiates PC12 into sympathetic ganglion chief neurons that lack the ability to synthesize GABA.³⁷

Table 1.

Summary of Protein Expression in AMC Cells and Unstimulated and NGF-Stimulated PC12 Cells.

		AMC	Unstimulated PC	Stimulated PC
Secretion-related proteins	CgA	++	+
	Syt7	++	+
	37 KDa-Syn	+	++
	35 KDa-Syn	–	–	+
Mitochondria	UCP	UCP3	UCP4
GABA signaling proteins	GAD67	+^a	+	–
	VGAT	+^a	+	–
	Gephyrin	+^b	+	–

Was obtained from 12, 33, and 44, whereas ^b was from 43.

UCP3 and UCP4, which are involved in energy metabolism in a cell-specific manner, were expressed in AMC cells and unstimulated PC12 cells, respectively, and glucocorticoid activity exerted an inhibitory effect on UCP4 expression. Chromaffin granule–associated proteins were minimally expressed in PC12 cells, whereas the amount of synaptophysin, an SLMV-associated protein, was much higher in PC12 cells compared with the adrenal medulla (Table 1). As found in AMC cells, M₁R was located at the cell periphery in unstimulated PC12 cells, whereas in NGF-stimulated PC12 cells, there was enhanced expression of M₁R at the cell periphery. The proteins involved in GABA auto/paracrine function in AMC cells were expressed in unstimulated PC12 cells, but not in NGF-stimulated cells. Based on the present and previous findings (Table 1), we conclude that the properties of PC12 cells under basal conditions are between those of AMC cells and sympathetic ganglion cells, and GABA signaling is intrinsic to AMC cells.

Footnotes

Competing Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Author Contributions

KH, HM, and MI contributed to the conception and design of the work; MI, KH, and HM generated the preparations; KH and HM acquired the data; KH and MI analyzed the data; MI wrote the manuscript. All authors have read and approved the final manuscript.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported, in part, by grants from JSPS KAKENHI (17K08555 to MI and 18K06865 to HM).

ORCID iD

Masumi Inoue

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