Nerve growth factor- and non-stimulated PC12 cells as models for sympathetic ganglion neurons and adrenal chromaffin cells

doi:10.21203/rs.3.rs-2217398/v1

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Nerve growth factor- and non-stimulated PC12 cells as models for sympathetic ganglion neurons and adrenal chromaffin cells

https://doi.org/10.21203/rs.3.rs-2217398/v1

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PC12 cells are an immortalized cell line originating from rat adrenal medullary chromaffin (AMC) cells and extend a neurite-like structure in response to nerve growth factor (NGF). Thus, non-stimulated and NGF-stimulated PC12 cells are used as model cells for AMC cells and sympathetic ganglion cells, respectively. However, how closely non-stimulated and stimulated PC12 cells resemble AMC cells and sympathetic neurons, respectively, has not sufficiently been elucidated. In addition, how the properties of PC12 cells are affected by NGF remains unclear. These issues were explored by using biochemical and immunocytochemical methods. AMC cells and PC12 cells selectively expressed UCP3 and UCP4, respectively, key proteins which are involved in energy metabolism in a cell-specific manner, and glucocorticoid activity exerted an inhibitory effect on UCP4 expression in PC12 cells. The expression levels of chromaffin granule-associated proteins in PC12 cells were extremely small, whereas the amount of synaptophysin, a synaptic vesicle-associated protein, was much larger than that in the adrenal medulla. As found in AMC cells, the muscarinic receptor subtype 1 (M₁R) were located at the cell periphery in basal PC12 cells, as was markedly enhanced by NGF. Furthermore, the proteins involved in GABA auto/paracrine function in AMC cells were expressed in non-stimulated PC12 cells, but not in NGF-stimulated cells. The results suggest that the properties of basal PC12 cells are between those of AMC cells and sympathetic ganglion cells, and glucocorticoid activity and NGF induce differentiation into AMC cells and sympathetic neurons, respectively, and GABA functions as an auto/paracrine factor in AMC cells.

PC12

chromaffin cell

chromogranin

synaptophysin

GAD67

UCP

Rat adrenal pheochromocytoma, PC12 cells, is an immortalized cell line originating from rat adrenal medullary chromaffin cells (AMC) cells [1]. This cell line has been used as a model cell to explore catecholamine secretion [2–3], membrane excitability [4], and hypoxia sensing in AMC cells [5]. Furthermore, because PC12 cells extend neurite-like structures in response to nerve growth factor (NGF), NGF-stimulated cells have also been used as a model cell for sympathetic ganglion cells [1, 6]. However, as far as we know, how NGF-stimulated and non-stimulated PC12 cells resemble sympathetic ganglion cells and AMC cells, respectively, has not sufficiently been elucidated.

AMC cells secrete catecholamine in response to neuronal and humoral inputs, functioning as endocrine cells [7]. Synaptic transmission from the sympathetic preganglionic nerve fibers to rat and guinea pig AMC cells is mediated by nicotinic and muscarinic receptors [8–11], whereas a decrease in blood pH is sensed by homomeric TASK1 channels in AMC cells with the consequent increase in catecholamine secretion [4, 12–14], thereby adapting the body to a metabolic stress occurring in strenuous exercise [15]. The secretary activity of AMC cells in response to synaptic and humoral inputs is assumed to be fine-tuned by the auto/paracrine function of GABA [16–19]. Whether the proteins involved in such signal transduction and regulatory mechanisms are expressed in PC12 cells or not is not sufficiently elucidated. If a group of proteins involved in a certain function cease to be expressed in NGF-induced differentiation, they could be assumed to contribute somehow to the endocrine function of AMC cells. Thus, the properties of non-stimulated and stimulated PC12 cells are helpful to understand the endocrine function of AMC cells. However, how the proteins change in response to NGF or glucocorticoids, which play an important role for maturation of AMC cells [2], remains unclear. Thus, the present experiment aims to address these issues by using the biochemical and immunocytochemical approaches.

Animals

Male rats and guinea pigs (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 light/dark cycle of 12/12 h. 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 [14]. Briefly, ten rats and ten 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 CaCl_2, 0.5 mM MgCl₂, 0.53 mM NaH₂PO₄, 5 mM D-glucose, and 5 mM Hepes, and 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 and the treatment was twice repeated in the 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, USA), 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 studies, cells were treated overnight with rabbit anti-uncoupling protein (UCP) 2 antibody (Ab) (AB3040; Chemicon, Temecula, CA, USA) (RRID: AB_2213932), rabbit anti-UCP3 Ab (Affinity Bioreagents, Golden, CO, USA), rabbit anti-UCP4 Ab (sc-20815; Santa Cruz Biotechnology, Dallas, TX, USA) (RRID: AB_2270488), or mouse anti-UCP4 Ab (sc-365295; Santa Cruz Biotechnology) (RRID: AB_10843244) [20]. After incubation, cells were washed three times with PBS and treated with a respective secondary Ab conjugated with Alexa Fluor 488 or 546 (Molecular Probes, Eugene, OR, USA). Fluorescence was observed under a laser confocal microscope (LSM5 Pascal: Carl Zeiss, Tokyo, Japan). The objective lens was an oil-immersion with a magnification of 63x and a numerical aperture of 1.4. For Alexa Fluor 488, a 514 nm laser was used, and 530 to 600 nm emission 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).

Immunoblotting

Five rats and five guinea pigs were killed by cervical dislocation and the adrenal glands and brains were excised and immediately put into ice-cold Ca²⁺-deficient balanced salt solution. The isolated adrenal medullae and brains were individually minced and homogenized with a Potter-Elvehjem homogeniser in 10 volumes of a solution containing 10 mM Tris-HCl (pH 7.4), 150 m NaCl, and a protease inhibitor cocktail (set 1: Calbiochem, San Diego, CA, USA). Homogenates were centrifuged at 500 g for 10 min at 4°C to remove the nuclei, then the postnucleus supernatants were mixed with equal volumes of an SDS 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, USA). After the addition of 2-mercaptoethanol (final concentration, 5% v/v) and bromophenol blue (0.05% v/v) to the samples, proteins (about 5 mg) 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) fat-free 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 (21–22), mouse anti-synaptophysin Ab (SY 38; Progen, Heidelberg, Germany) (RRID: AB_1543003), rabbit anti-dopamine b-hydroxylase (DbH) Ab (AB1538; Chemicon) (RRID: AB_90751), rabbit vesicular GABA transporter (VGAT) Ab (VGAT11-s; Alpha Diagnostic, San Antonio, TX, USA) (RRID: AB_2190109) [23], mouse anti-gephrin Ab (sc-25311; Santa Cruz Biotechnology) (RRID: AB_627670) [24], mouse anti-actin Ab (MAB1501R; Sigma-Aldrich) (RRID: AB_2223041), or mouse anti-glutamic acid decarboxylase (GAD) 65/67 Ab (73–508; NeuroMab, Davis, CA, USA) (RRID: AB_2756510), or rabbit anti-UCP3 Ab (Affinity Bioreagents). The immunoreaction was detected by incubating the membrane with a secondary Ab linked to horseradish peroxidase (Amersham, Buckinghamshire, UK), and then with ECL-Plus (Amersham).

Cell Culture

PC12 cells (RRID: CVCL_0481) originating from male rat adrenal medullary cells [1] were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco: Life Technologies, Tokyo, Japan) supplemented with 10% fetal FBS. INS-1 cells (RRID: CVCL_0352), a cell line originating from rat insulin-secreting cells [25], were cultured in RPM1 1640 (Gibco) supplemented with 10% FBS, 10 mM Hepes, 11.1% glucose, 1 mM sodium pyruvate, and 50 mM 2-mercaptoethanol. For immunostaining, the cells were placed onto glass coverslips coated with collagen type I (BD Biosciences, San Jose, CA, USA). The cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After washing three times with PBS, 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 receptor 1 (M₁R) Ab (Frontier Institute, Ishikari, Japan) (RRID: AB_2571791), mouse anti-M₂R Ab (sc-71531; Santa Cruz Biotechnology) (RRID: AB_1126414), mouse anti-M₄R Ab (MAB1576; Chemicon) (RRID: AB_2080217), or rabbit anti-M₅R Ab (6391 − 2190; Biogenesis, Poole, UK) and then with secondary Abs. The specificities of the anti-muscarinic receptors Abs were previously confirmed in our hands (Harada et al., 2011). For NGF stimulation, the cells were serum-starved for 12 h before application of 50 ng ml⁻ NGF and cultured for one week in DMEM with (high K⁺ medium) or without addition of 20 mM KCl [26]. For immunoblotting, the PC12 cells were cultured in 100 mm dishes, and the stimulated and non-stimulated 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 xg for 30 min at 4°C. The cell lysate of the supernatant was added to an equal volume of the SDS buffer.

RT-PCR

The expression of UCPs at the mRNA level was examined with a reverse transcriptase (RT)-PCR approach, as described elsewhere [9]. Briefly, the Micro-fast track kit (Invitrogen: Life Technologies) was employed to isolate poly(A)⁺RNA from the brains, adrenal medullae, and adrenal cortices of five rats according to the manufacturer’s instructions. Oligo dT primer was utilized for the RT reaction to obtain cDNAs. PCR reactions were carried out with 1.25 ml of DNA template, 4 pmole of primer, 2 mM of dNTPs, 0.5 units of rTag (Takara, Otsu, Japan), and PCR buffer supplied with the kit in a final volume of 20 ml. The PCR protocol consisted of an initial 3 min denaturation step at 94°C and the subsequent 30–40 cycles of the profile comprising 30 s of denaturation at 94°C, 30 s of annealing at 54 to 60°C and 30 s of extension at 72°C. PCR products were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide.

Sources Of Reagents

Collagenase was obtained from Yakult (Kunitachi, Japan); a rabbit anti-synaptotagmin 7 Ab was kindly provided by Dr. M. Fukuda (Tohoku University, Sendai, Japan).

Statistics

Data were expressed as means ± SEM. The data with a normal distribution (Shapiro-Wilk test) was assessed with unpaired Student’s t-test or Bonferroni t-test. Otherwise, Mann-Whitney rank sum test or Kruskal-Wallis analysis of variance on ranks test was used. A p-value < 0.05 defined a statistically significant difference. Statistical analysis was performed with Sigma Plot v13.0 software (Systat Software, San Jose, CA, USA).

Secretion-related proteins

Immunoblotting was used to explore how much PC12 cells succeed to the properties of original rat AMC cells. The expression level of 70-kDa chromogranin A, a major protein constituting chromaffin granules [27], was extremely low in PC12 cells, compared with that in the rat adrenal medulla (Fig. 1A). In parallel to a low expression of chromogranin A, 40 and 43 KDa bands of synaptotagmin 7 [21, 28], a major Ca²⁺ sensor in catecholamine-storing chromaffin granules [22, 29], 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 level of 38-kDa synaptophysin, a marker protein for synaptic vesicles or synaptic-like microvesicles (SLMVs) [30–31], in PC12 cells was five times larger than that in the adrenal medulla homogenates (Fig. 1C). These results raise the possibility that the significant fraction of secretary activity in PC12 cells occurs through SLMVs rather than through large dense core vesicles (LDCVs) [32]. Consistent with this notion, 83-KDa dopamine b-hydroxylase (DbH) [33], an enzyme mediating biosynthesis of noradrenaline from dopamine was scarcely detected in PC12 cells homogenates, compared with that in the adrenal medulla (Fig. 1D). Lastly, the molecular form of synaptophysin was investigated. As is known [34], the apparent molecular weight of synaptophysin in the brain was 34 KDa, whereas that in the adrenal medulla and the PC homogenates was 38 KDa because of glycosylation (Fig. 1E). When PC12 cells were stimulated by NGF for one week, 29.6 ± 2.7% (n = 3) of the synaptophysin molecules were found to exist as a 34 KDa form, suggesting that part of NGF-stimulated PC12 cells differentiate into neurons.

UCP

UCP is in the inner membrane of mitochondria and plays an important role for energy metabolism [35–36]. UCP comprises five isoforms, which exhibit a selective expression in different tissues [37]. Therefore, UCP isoforms expressed in AMC and PC12 cells were immunocytochemically explored (Fig. 2A). As reported previously [38], UCP2-like immunoreactive (IR) material was detected in the cytoplasm in INS-1 cells deriving from rat pancreatic b cells [25] (not shown), indicating that the anti-UCP2 Ab used was immunoactive. This Ab did not generate immunofluorescence in the cytoplasm in guinea pig AMC (n = 8), rat AMC (n = 5), or PC12 cells (n = 3). In contrast to UCP2, UCP3-like IR material was detected in the cytoplasm in guinea pig (n = 17) and rat AMC cells (n = 11), but not in PC12 cells (n = 12). The expression of UCP4 was examined with two different anti-UCP4 Abs. The treatment with a rabbit anti-UCP4 Ab produced UCP4-like IR material in the cytoplasm in PC12 cells (n = 10), but not in guinea pig (n = 13) or rat AMC cells (n = 11). These results were reproduced by a monoclonal anti-UCP4 Ab (Fig. 3), which has been confirmed to be specific to UCP4 [20].

The expression of UCP3 in AMC cells was further examined by using immunoblotting. As is evident in Fig. 2B, a 32 KDa band [39] was detected in each homogenate of guinea pig adrenal medulla, adrenal cortex and brain, and the same result was obtained in the rat samples (not shown). The expression of UCP isoforms at the mRNA level was next examined using an RT-PCR approach. Figure 2C shows that PCR products for UCP2 were amplified in the rat adrenal cortex, but not in the brain or adrenal medulla samples, whereas those for UCP3 were recognized in all the adrenal cortex, adrenal medulla, and brain samples. As expected [37], UCP4 PCR products were amplified in the brain samples, but not in the adrenal medulla or adrenal cortex. The results indicate that UCP3 and UCP4 are selectively expressed in AMC and PC12 cells, respectively.

Higher concentrations of glucocorticoids to which AMC cells in vivo are exposed play an important role for the development of AMC cells [40]. Thus, we investigated the effects of dexamethasone, a synthetic glucocorticoid, on the expression of UCP4 in PC12 cells. As is evident in Fig. 3, A and B, the amount of UCP4-like IR material in the cytoplasm diminished in a time-dependent manner in the presence of dexamethasone. When PC12 cells were cultured in a medium containing 5 mM dexamethasone for one week, the level of UCP4-like immunofluorescence in the cytoplasm decreased by 44.5%.

Muscarinic Receptor Subtypes

The synaptic transmission from sympathetic preganglionic nerve fibers to the AMC cells is in part mediated by muscarinic receptors [7–9]. The M₁R has been found to produce a depolarization through the inhibition of TASK1 channels in rat AMC cells [4, 13], whereas the M₁R and M₄R mediate M-type K⁺ channel [41] and voltage-dependent Ca²⁺ channel inhibition [41–42] in rat sympathetic ganglion cells, respectively. Thus, the expression of muscarinic receptor subtypes was immunocytochemically explored by using isoform-specific Abs [9]. NGF stimulation has been reported to result in a decrease in M₁R expression [43]. However, immunoblotting clearly showed that the amount of 64-kDa M₁R band [44] in NGF-simulated PC12 ells was significantly larger than that in non-stimulated cells (Fig. 4, A and B). This enhancement of expression was further facilitated in the presence of high K⁺ ions, where NGF-induced differentiation into sympathetic neurons is facilitated [26, 45]. Immunocytochemistry evidently revealed that part of M₁R-like IR material was located at the cell periphery in non-stimulated and NGF-stimulated cells (Fig. 4, C and D). This distribution tended to be enhanced in the presence of 20 mM K⁺ (Fig. 4, E and F). As previously reported [43], M₂R- and M₄R-like materials also increased with NGF stimulation and were preferentially located in dot-like manner at the cell periphery (Fig. 4, C, D, and F). In contrast to M₁R, M₂R, and M₄R, the majority of M₅R-like IR material was in the cytoplasm in a reticular manner (Fig. 4D), suggesting the presence of M5R-like IR material in the endoplasmic reticulum [46].

Proteins Involved In Gaba Signaling

Our previous experiment [18] revealed that GAD67, one of the GABA synthesizing enzymes (GAD65 and GAD67) [47–48], is expressed at the mRNA level in PC12 cells. Consistent with this result, a 67-KDa GAD band was detected in the homogenates of non-stimulated PC12 cells (Fig. 5, A and B). Intriguingly, this band was absent in the homogenates obtained from NGF- or dexamethasone-stimulated PC12 cells. Similarly, 50-KDa VGAT [49], an enzyme involved in uptake of GABA into secretary vesicles, was detected in the non-stimulated PC12 homogenates, but not in the NGF-stimulated PC12 (Fig. 5, C and D). In contrast to GAD, the amount of VGAT in PC12 cells was maintained in dexamethasone-stimulated PC12 cells. Gephrin with an apparent molecular weight of 98 KDa [50], one of the proteins anchoring GABA_ARs to the subsynaptic membrane in the brain [51–52], was detected in the non-stimulated PC12 homogenates. However, NGF stimulation abolished the expression of gephrin in PC12 cells, and the expression level of gephrin in PC12 cells was not maintained by dexamethasone stimulation.

NGF-induced differentiation

The survival of the sympathetic ganglion neurons 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 [53], and NGF facilitates the extension of neurites in PC12 cells, suggesting that NGF induces PC12 cells to differentiate into sympathetic ganglion cells. This notion is supported by the fact that AMC cells and sympathetic ganglion cells are developmentally close [54]. NGF-differentiated PC12 cells, however, produce choline acetyltransferase (ChAT) with the consequent generation of ACh as a neurotransmitter [55–56]. This fact may not negate the differentiation into sympathetic ganglion cells in response to NGF. The reason is that sympathetic ganglion neurons that are cultured with cardiac myocytes are known to produce ACh as a neurotransmitter [57–58] and sympathetic ganglion neurons innervating the sweat gland switches a neurotransmitter from noradrenaline to ACh [45]. Thus, sympathetic ganglion cells themselves are plastic on neurotransmitters.

M₁R activation in sympathetic ganglion cells results in the inhibition of M-type K⁺ channels with the consequent depolarization [41], whereas M₄R stimulation produces the suppression of voltage-dependent Ca²⁺ channels through a membrane-delimited manner [41–42]. Consistent with these findings, NGF stimulation produced an increase in membrane expression of M₁R and M₄R in PC12 cells. Selective localization of M₁Rs at the cell periphery was conspicuously enhanced by NGF in the presence of high K⁺ ions, which is known to facilitate differentiation into catecholaminergic neurons [58]. However, the enhanced expression of M₁R in response to NGF is opposite to the previous report where NGF stimulation was concluded to upregulate M₂R and M₄R and downregulate M₁R in PC12 cells [43]. This contradiction may be ascribed to a difference in the Abs used in the current and previous experiments. In both experiments, different commercially available Abs were used, and the immunoreactivity of the current anti-M₁R Ab was confirmed in our hands [9].

It would be worth noting that M₁R mediates the slow excitatory postsynaptic potential in sympathetic ganglion neurons of various animals [59], whereas the involvement of muscarinic receptors in the synaptic transmission in AMC cells is variable, depending on the animal species [9, 11, 60]. In rat AMC cells, muscarinic receptor-mediated transmission may occur under the severe stressful conditions [9, 60], whereas such a restriction may not be the case for guinea pig AMC cells [11]. The finding that M₁R expression in PC12 cells was enhanced under conditions where the differentiation into catecholaminergic cells is facilitated might suggest that in the rat M₁R-mediated synaptic transmission plays more important role in sympathetic neurons than in AMC cells.

Model As Amc Cell

The present experiment clearly revealed that the expression levels of the proteins involved in catecholamine synthesis, storage, and secretion were conspicuously low, compared with those in AMC cells. This fact may not be due to immaturity of PC12 cells as endocrine cells. The expression level of synaptophysin, a marker protein for the synaptic vesicle or SLMV [30, 34], was five time larger than that in AMC cells. However, the molecular form of synaptophysin in non-stimulated PC12 cells was not of the neuron-type [34], and NGF stimulation facilitated its expression in the neuronal form in PC12 cells. The results suggest that non-stimulated PC12 cells have the properties between AMC cells and sympathetic ganglion cells. This notion is supported by a different expression of UCP isoforms in AMC cells and PC12 cells.

The UCP family consists of five isoforms [37]: UCP1 is mainly detected in BAT (brown adipose tissue) and involved in thermogenesis; UCP2 is present in various types of cells, such as endocrine cells and lymphocytes [37, 61]; UCP3 is expressed in skeletal muscles and adult cardiac myocytes and involved in transport of fatty acids in mitochondria [62]. There is a close relationship between its expression level and the extent of fatty acid b-oxidation [63]. UCP4 and UCP5 are selectively expressed in neurons [64–65]. Immunocytochemistry with an anti-UCP3 Ab whose specificity was confirmed in our hands revealed UCP3-like IR material in AMC cells and not in PC12 cells. On the other hand, immunocytochemistry with two different anti-UCP4 Abs showed UCP4-like IR material in PC12 cells and not in AMC cells. These immunocytochemical findings are consistent with those obtained with RT-PCR analyses. Thus, we conclude that UCP3 and UCP4 are selectively expressed in AMC cells and PC12 cells, respectively. It would be worth considering the physiological significance for UCP3 expression in AMC cells. This isoform is assumed to be involved in fatty acid transport in mitochondria [63] with the consequent increase in oxidative phosphorylation. Thus, UCP3 expression helps 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.

The intermediate properties of PC12 cells are further supported by the expression of proteins involved in GABA signaling in non-stimulated PC12 cells. Immunoblotting revealed that VGAT, GAD67, and gephrin were expressed in non-stimulated PC12 cells. Our [23, 66] and other research [67] 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, gephrin is the major protein mediating anchoring of GABA_ARs to the subsynaptic membrane in brain neurons [51–52]. This scaffolding protein was not expressed in rat AMC cells [24]. Thus, it is not clear why gephrin is expressed in non-stimulated PC12 cells. It, however, would be worth to note that PC12 cells express not only a3, but also a1 subunits of the GABA_ARs [24]. The a1 is the major a subunit of GABA_ARs mediating the inhibitory postsynaptic potential (IPSP) in adult brain neurons [68], whereas the a3 is one of the a subunits constituting GABA_ARs involved in IPSPs in the neonatal period [68]. It has been known that the a3 subunit in the GABAergic subsynaptic membrane in a certain brain area is replaced with the a1 in the brain development [69–71]. Culturing PC12 cells in the presence of dexamethasone resulted in a decrease in a1 expression and an increase in a3 [24]. Interestingly, in the presence of dexamethasone VGAT expression was maintained, but gephrin was not. Because the a1 subunit has a binding site for gephrin [72], the results might suggest that the expression of the a1 is somehow coupled with that of gephrin with the consequent facilitation of inhibitory synaptic transmission. The findings that any of VGAT, GAD67, and gephrin were not expressed in NGF-stimulated PC12 cells further supports that NGF differentiates PC12 into sympathetic ganglion chief neurons that lack GABA synthesizing capability [73] and GABA functions as an auto/paracrine factor in AMC cells.

Intriguingly, the expression of UCP3, one of the neuronal isoforms [74], in PC12 cells was suppressed by culturing in the presence of dexamethasone, as was noted for the expression of GABA_AR a1 subunit [24]. Glucocorticoids are known to suppress neurite extension of NGF-stimulated PC12 cells [75]. These results indicate that glucocorticoid activity is involved in the suppression of differentiation into a neuron. Thus, exposure to high concentrations of glucocorticoids is necessary for AMC cells to function as an endocrine cell secreting catecholamines.

Finally, it is worth discussing the appropriateness of using PC12 cells to explore the O₂-sensing mechanism in AMC cells. ACM cells in the perinatal period secrete catecholamine in response to hypoxia [76–78]. This secretion results from detection of hypoxia by AMC cells themselves. Thus, the O₂-sensing mechanism in AMC cells has been subjected to intensive investigation for a long period [79], and PC12 cells have been used as a model of AMC cells [5]. The present experiment reveals that isoforms of UCP that are crucially involved in cellular metabolism [35] are different between PC12 cells and AMC cells, suggesting that PC12 cells are not an ideal preparation for studies of the O₂-sensing mechanism in AMC cells.

UCP3 and UCP4, which are involved in energy metabolism in a cell-specific manner, were expressed in AMC cells and non-stimulated PC12 cells, respectively. The expression levels of chromaffin granule-associated proteins in PC12 cells were extremely low, compared with those in the adrenal medulla. The amount of synaptophysin, a SLMV-associated protein, in PC12 cells was much more than that in the adrenal medulla. As found in AMC cells, M₁R was located at the cell periphery in non-stimulated PC12 cells, whereas in NGF-stimulated PC12 cells the expression of M₁R at the cell periphery was enhanced. Furthermore, the proteins involved in GABA auto/paracrine function in AMC cells were expressed in non-stimulated PC12 cells, but not in NGF-stimulated cells. These results suggest that the properties of PC12 cells under basal conditions are between those of AMC cells and sympathetic ganglion cells. These characteristics should be taken into account when PC12 cells are used a substitute for AMC cells.

Competing Interests

The authors have no conflicts of interest to declare.

Funding

This study was supported, in part, by grants of JSPS KAKENHI (17K08555 to M. I. and 18K06865 to H. M.).

Author Contributions

K. H., H. M., and M. I. contributed to the conception and design of the work; M.I., K. H., and H. M. made the preparation; K. H. and H. M. acquired the data; K. H. and K. H. analyzed the data; M. I. wrote the manuscript. All authors have read and approved the final manuscript.

Data Availability

The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.

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Nerve growth factor- and non-stimulated PC12 cells as models for sympathetic ganglion neurons and adrenal chromaffin cells

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Abstract

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Introduction

Materials And Methods

Animals

Immunocytochemistry

Immunoblotting

Cell Culture

RT-PCR

Sources Of Reagents

Statistics

Results

Secretion-related proteins

UCP

Muscarinic Receptor Subtypes

Proteins Involved In Gaba Signaling

Discussion

NGF-induced differentiation

Model As Amc Cell

Conclusion

Declarations

References

Additional Declarations

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