The Inhibition Effects of Shenmai Injection on Acetylcholine-Induced Catecholamine Synthesis and Secretion by Modulating Nicotinic Acetylcholine Receptor Ion Channels in Cultured Bovine Adrenal Medullary Cells

Shenmai injection (SMI) has been widely used for the treatment of cardiovascular diseases in China. Cardiovascular disorders are often related to excessive catecholamine (CA) secretion. Here, we report the effects of SMI on CA secretion and synthesis in cultured bovine adrenal medullary cells. We found that SMI significantly reduced CA secretion induced by 300 μM acetylcholine (ACh). Cotreatment with SMI (10 μL/mL) and either of the ACh receptor α-subunit inhibitors, HEX (α3) or DhβE (α4β2), did not produce any further inhibition, indicating that SMI may play a role through α3 and α4β2 channels. Furthermore, SMI reduced tyrosine hydroxylase (TH) activity induced by ACh by inhibiting the phosphorylation of TH at Ser19 and Ser40. TH is phosphorylated at Ser19 by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and at Ser40 by protein kinase A (PKA). KN-93 and H89, the antagonists of CaM kinase II and PKA, respectively, inhibited the ACh-induced phosphorylation at Ser19 and Ser40, and the addition of SMI did not augment the inhibitory effect. Taken together, our results show that SMI likely inhibits CA secretion by blocking TH activity at its Ser19 and Ser40 sites.


Introduction
Catecholamines (CAs) are a class of neurotransmitters including norepinephrine (NE), epinephrine (E), and dopamine (DA). CAs regulate normal cardiovascular functions at physiological levels, while excessive secretion can aggravate cardiovascular diseases such as heart failure, atherosclerosis, coronary heart disease, hypertension, and so on [1].
Bovine adrenal chromaffin cells, also called adrenal medullary cells, were derived from embryonic neural crests with homologous function in sympathetic neurons [2]. Hence, bovine adrenal medullary cells were widely used to detect the effect of various medicines or chemicals on catecholamine secretion and synthesis. Researchers found that there are at least three distinct types of ionic channels that participate in catecholamine secretion, including nicotinic acetylcholine receptor (nAChR) ion channels, voltage-dependent Na + channels, and voltage-dependent Ca 2+ channels [3]. In cultured bovine adrenal medullary cells, Ach and veratridine were reported to be activators of the nicotinic acetylcholine receptor and voltage-dependent Na + channels, respectively. Both ACh and veratridine caused Na + influx and depolarization of membrane after they acted with the above receptors [4]. en, depolarization of membrane could cause the opening of voltage-dependent Ca 2+ channels. Besides, high K + (HK) directly activated voltage-dependent Ca 2+ channels to increase Ca 2+ influx without increasing Na + influx [5]. Ca 2+ influx is the essential trigger for CA synthesis and secretion [6]. It was found that the final raise of introcellular (Ca 2+ ) induced by ACh, Ver, and HK induced the secretion of catecholamine. On the other hand, it was known that biosynthesis of CA began with dihydroxyphenylalanine (DOPA) which was converted to dopamine (DA); the latter was then modified into norepinephrine (NE) and epinephrine (E) [7]. Tyrosine hydroxylase (TH), which catalyzed the rate-limiting step in catecholamine biosynthesis [8], was activated via phosphorylation at the Ser19, Ser31, and Ser40 sites in correspondence to following multiple protein kinases, including Ca 2+ /calmodulin-dependent protein kinase II (CaM kinase II), extracellular signal-regulated protein kinase (ERK), and cAMP-dependent protein kinase A (PKA) [9,10].
Although toxicity studies have confirmed the general safety of SMI [18,19], its effects and mechanisms on catecholamine secretion and synthesis have not been fully investigated.
erefore, in the current study, bovine adrenal medullary cells were used to investigate the effect and the potential mechanisms of SMI on CA synthesis and secretion. First, we primary cultured the bovine adrenal medullary cells and established the ACh, Ver, and HK-induced CA secretion model to investigate the effect of SMI on CA secretion by using HPLC coupled with the electrochemical detection (HPLC-ECD) method and introcellular Ca 2+ by an inverted fluorescent microscope. en, we used the inhibitors of corresponding subunits of nAChR to confirm in which subunits of the channels did SMI exerts its effects on inhibiting Ca 2+ influx and CA secretion. Finally, we detected the effects of SMI on the activity of TH and its possible phosphorylation sites to investigate the effect and mechanism on CA synthesis. e cell culture reagents trypsin inhibitor, collagenase L, and penicillin-streptomycin and collagenase type 1 were purchased, respectively, from Nacalai Tesque (Kyoto, Japan), Wako (Kyushu, Japan), and Gibco (California, CA, USA). Finally, the TH activity detection kit was purchased from Genmed (Shanghai, China) and Fluo4-AM from Dojindo (Kyushu, Japan).

Isolation and Primary
Culture of Bovine Adrenal Medullary Cells. Bovine adrenal glands were obtained from the City Slaughter House. e chromaffin cells were isolated from the adrenal glands by collagenase digestion as described previously [20]. e cells were cultured at a density of 5 × 10 5 cells/well in Eagle's MEM supplemented with 10% FCS, 50 U/mL penicillin, and 50 g/mL amphotericin B under a humidified atmosphere of 5% CO 2 and 95% air at 37°C in 12-well, 24-well, and 48-well plates. Primary cells harvested between 2 and 5 days of culture were used for experiments.

Catecholamine Secretion from Cultured Bovine Adrenal Medullary Cells.
To detect the effect of SMI on CA secretion, the level of CAs in culture medium was detected by the highperformance liquid chromatographic system coupled with an electrochemical detector (HPLC-ECD). Briefly, bovine adrenal medullary cells were buffered with oxygenated KRP and washed 3 times before experiments. After incubation with the indicated agents at 37°C for 10 min, the medium was transferred immediately to a test tube containing perchloric acid (PCA) (final concentration, 0.4 M), and NE and E secreted into the medium were detected by HPLC-ECD. e chromatographic separation system included a RP-C18 analytical column (5 μm, φ4.6 mm × 250 mm) and an isocratic solvent system. e flow phase (1 L) contained 100 mL acetonitrile, 100 μL triethylamine, 13.6 g KH 2 PO 4 , 2.5 g octane-sulfonic acid sodium, and 0.036 g EDTA, pH adjusted to 3 with H 3 PO 4 . e flow rate was 0.6 mL/min, column temperature was 30°C, and sample injection volume was 10 μL. An electrochemical detector with two channels (0 mV and +300 mV) was used to detect the signals. Evidence-Based Complementary and Alternative Medicine Chromaffin cells were buffered with Ca 2+ KRP and washed 3 times before experiments. After 10 min preincubation with different dilutions of SMI, cells were incubated for an additional 10 min in the presence or absence of various secretagogues. e incubation medium was then transferred immediately to a test tube containing perchloric acid (PCA) (final concentration, 0.4 M). NE and E secreted into the medium were directly injected and detected by HPLC-ECD as described above.

Measurement of Intracellular Ca 2+ Mobilization.
To examine the effects of SMI on intracellular Ca 2+ concentration induced by 300 μM ACh, cells were seeded into black-walled clear-base 96-well plates (CORING, USA) in culture media and cultured for approximately 24 h in a 37°C CO 2 incubator. e culture media was removed, and the cells were washed with KRP buffer three times. e cells were then incubated with 2 μM Fluo4-AM for 20 min at 37°C and then washed three times with Ca 2+ (+) KRP buffer. After a final incubation in KRP buffer for 20 min at 37°C, changes in fluorescence induced by the Fluo4 complex was measured at an excitation wavelength of 494 nm and an emission wavelength of 516 nm before and after the addition of SMI (0.25-1 μL/mL), with a microplate reader (SpectraMax 190, Molecular Devices, USA), and the cells were observed by an inverted fluorescent microscope (DM750 M, LEICA, Germany) at 20x amplified.

In Situ Detection of TH Activity.
e cells cultured at a density of approximately 1 × 10 7 cells per well were washed three times with PBS buffer. en, the cells were incubated with the TH activity detection kit according to the manuscript's instructions. e TH activity was measured in terms of fluorescence (ex � 280 nm, em � 320 nm) by a microplate reader.

In Situ Phosphorylation of TH.
e effect of SMI on the inhibition of phospho-Ser19, phospho-Ser31, and phospho-Ser40 of TH was assayed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Cells (2 × 10 6 /dish) were incubated with or without SMI (0.25-1 μL/mL) for 20 min in KRP buffer. e cells were washed twice with ice-cold KRP buffer, lysed in lysis buffer, and the lysates were centrifuged at 15,000x g for 30 min.
Protein concentration was measured using an enhanced BCA Protein Assay Kit, and equal amounts of protein were mixed with 5x SDS-PAGE sample loading buffer. Following heating at 95°C for 5 min, proteins were subjected to 10% Tris-glycine gel SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membranes (BioRad). e membranes were incubated with the primary antibodies (1 : 1000) overnight at 4°C and then washed with phosphatebuffered saline containing 0.1% Tween 20 (PBS-T), followed by incubation with the rabbit radish-peroxidase-labeled secondary antibody (1 : 10000) at room temperature for 1 h. e protein bands were visualized using enhanced chemiluminescence by Western blot detection reagents, and the band densities were measured by Quantity One software using the Versa Doc imaging system (BioRad). Data were expressed as the ratio of phosphorylated proteins to total proteins.

Statistical Analysis.
All experiments were performed in triplicates, and each experiment was repeated at least 3 times. e data were presented as mean ± SEM. Statistical significance was evaluated using the t-test or one-way analysis of variance (ANOVA), and p < 0.05 was considered significant. All statistical analyses were performed using SPSS 11.5 software (SPSS Inc., Chicago, IL, USA).

Effect of SMI on Basal and Secretagogue-Induced CA Secretion in Cultured Bovine Adrenal Medullary Cells.
To study the effect of SMI on CA secretion, bovine adrenal medullary cells were treated with various secretagogues in the presence or absence of SMI (2.5 μL/mL). SMI did not affect the basal CA secretion in the control group that treated with no secretagogue (p > 0.05). Upon cotreatment with different secretagogues for 10 min, 2.5 μg/mL SMI significantly inhibited CA secretion induced by 300 μM ACh (p < 0.05) and caused a slight decline in 56 mM K + and 100 mΜ Ver-induced secretion (p > 0.05) (Figure 1(a)). We also examined the effect of SMI on CA secretion induced by 300 μM ACh. Cells treated with 2.5, 5, and 10 μL/mL SMI for 10 min showed significantly reduced ACh-induced secretion of CA (p < 0.05 and p < 0.01) compared to Ach-treated cells only (Figure 1(b)).

Inhibitory Role of SMI on Catecholamine Secretion Induced by 300 μM ACh.
e nAChR ion channels consist of three important α-subunit sites-α3, α4β2, and α7. To examine the involvement of voltage-dependent nAChR ion channels on the SMI effect, we treated the cells with specific inhibitors against the α-subunit, namely, HEX (α3), DhβE (α4β2), and MLA (α7).
After 10 min pretreatment with (or without) SMI (10 μL/ mL), cells were cotreated with 300 μM ACh and HEX (10 μM), DhβE (10 μM), or MLA (10 μM) for 10 min at 37°C; suitable untreated controls were included. As shown in Figure 2, HEX, DhβE, and MLA each inhibited ACh-induced CA secretion (p < 0.05). e inhibitory effect of MLA was further augmented in the presence of SMI (p < 0.05), while HEX and DHβE did not further increase the inhibitory effects of SMI.

Inhibition of SMI upon Increased Ca 2+ Influx Induced by 300 μM Ach in Bovine Adrenal Medullary Cells.
In order to further validate the inhibitory effect of SMI on nAChR ion channels, different concentrations of SMI (2.5, 5, and 10 μL/ mL) were used to increase Ca 2+ influx induced by 300 μM ACh, and the intracellular Ca 2+ concentrations were measured. SMI significantly suppressed the ACh-induced Ca 2+ influx, which is consistent with the results of CA secretion (Figure 3).

Effects of SMI on ACh-Induced Activity of Tyrosine
Hydroxylase. We next examined the effect of SMI treatment on ACh-induced TH activity. Cells treated with varying concentrations of SMI (2.5, 5, and 10 μL/mL) for 10 min at 37°C significantly reduced TH activity induced by ACh (p < 0.01) (Figure 4).

e Influence of SMI on TH Phosphorylation.
To determine which phosphorylated site(s) are affected by SMI, the effect of SMI on TH phosphorylation at the Ser19, Ser31, and Ser40 sites was examined. As shown in Figures 5(b) and 5(d), cells incubated with SMI (2.5, 5, and 10 μL/mL) for 20 min at 37°C showed a significant decrease in phosphorylation at Ser19 and Ser40 (p < 0.05, p < 0.01), but not at Ser31 (Figure 5(c)). TH is phosphorylated at Ser19 by CaM kinase II and at Ser40 by PKA. To determine the possible mechanism of the antiphosphorylation effect of SMI, cells were treated with 10 μM KN-93, a CaM kinase II antagonist, and 10 μM H89, a PKA antagonist. While both antagonists significantly suppressed the stimulatory effect of 300 μM ACh on TH activity (p < 0.05), addition of SMI (10 μL/mL) did not augment the inhibitory effect of either KN-93 or H89 (Figures 5(g) and 5(h)).

Discussion
In the present study, we found that SMI reduced the CA secretion induced by ACh in adrenal medullary cells, while it had no effect on veratridine and HK-induced catecholamine secretion. We also found that all the three doses of SMI (2.5 μL/mL, 5 μL/mL, and 10 μL/mL) inhibited ACh-induced Ca 2+ influx, a prerequisite for the secretion and synthesis of CAs, which was consistent with the results of CA secretion. To the best of our knowledge, this is the first time that SMI mediated inhibition of the function of nAChR ion channels and CA secretion in cultured bovine adrenal medullary cells.
Nicotinic ACh receptor (nAChR), as an ionotropic receptor, was widely reported to be involved in catecholamine secretion and synthesis [21]. Because of its important role in regulation of the nerve system and involvement in a variety of diseases, nAChR was well investigated for decades from its structure to the physical functions. It was reported that α3, α4β2, and α7 as nAChR ion channels important α-subunit sites play an important role on CA secretion. e most relevant nAChR for CA secretion is the heteromeric one composed of α3 and β4 subunits [22]. e α4β2 nAChR X-ray structure, recently elucidated, because of its heteromeric composition deserves further discussion [23]. A typical α7 agonist like choline was shown to stimulate  Evidence-Based Complementary and Alternative Medicine nAChRs on chromaffin cells to induce catecholamine secretion [24]. Ginsenoside Rg3, a chemical from Panax ginseng, was found to be voltage-dependent to inhibit AChinduced peak inward currents by α3β2, α3β4, α4β2, and α4β4 subunits. However, the same Rg3 effect on α7 nAChR channel activity was not observed [25]. It was reported that ginsenoside Rg2, a chemical in SMI, had been shown to block the nicotinic acetylcholine receptors in bovine chromaffin cells by affecting the acetylcholine-(Ach-) induced currents in heteromeric receptors α3β4, α3β2, α4β4, and α4β2, without affecting the acetylcholine-(Ach-) induced currents in α7 human receptors [26]. In the present study, we also found that cotreatment with SMI and the AChR α7 subunit antagonist MLA significantly augmented the inhibition of ACh-induced CA secretion compared to SMI treatment alone, whereas the combination of SMI with α3 and α4β2 antagonists HEX and DhβE did not produce any further inhibition. ese data indicated that SMI attenuates ACh-induced CA secretion potentially by preferentially inhibiting α3 and α4β2 channels with no effect on α7 channel. ese results were consistent with the findings in the previous reports on ginsenosides Rg2 and Rg3. Except for the catecholamine secretion, chromaffin cells are also an important method for the catecholamine synthesis study [27]. In our present study, we found that SMI significantly stimulated ACh-induced catecholamine synthesis indicating a possible mechanistic basis of reducing CA secretion. en, the mechanisms were also detected by testing the effect of SMI on tyrosine hydroxylase (TH) activity. In bovine adrenal medullary cells, TH catalyzes the conversion of tyrosine to L-3,4-dihydroxyphenylalanine (DOPA), the rate-limiting step in CA biosynthesis. erefore, TH activity is essential for the synthesis of catecholamine. In the present study, SMI inhibited the ACh-induced TH activity. It was reported that TH was regulated by an allosteric activation-induced shortterm regulation and an enzyme induction-induced longterm regulation. TH activity is acutely regulated by various modifications such as phosphorylation [28]. Phosphorylation at Ser40 in TH was essential to active the enzyme, while Ser19 phosphorylation had no direct effect on TH activity, but it increases TH activity indirectly by increasing the rate of phosphorylation at Ser40 [29]. So, clarifying the effect of SMI on phosphorylation of TH was important to demonstrate the stimulation of catecholamine synthesis and the TH activity. We found that SMI reduced the levels of phopho-Ser19 following ACh induction, which may likely decrease the phosphorylation rate of Ser40 and eventually decrease TH activity. In this study, KN-93 (an

Phospho-ser40TH
Total TH cardiovascular diseases, it is worth investigating the effect of SMI on the secretory CA system of adrenal medullary cells. CA neurotransmitters such as dopamine (DA), epinephrine (E), and norepinephrine (NE) have vital physiological roles. NE and E act as central nervous system stimulants resulting in faster heart rate, enhanced myocardial contraction, increased cardiac output, elevated blood pressure, reduced splanchnic blood flow, and increased adipose decomposition and oxidation. Stress is one of the reasons for the increased secretion of calcium synthetic hormones, and chronic stress can trigger abnormal immune functions, high blood pressure, coronary heart disease, and atherosclerosis. erefore, controlling the synthesis and secretion of CA through drug interventions has immense therapeutic potential. Ca 2+ influx is the essential trigger for CA synthesis and secretion, and nicotinic acetylcholine receptor (nAChR) ion channels play an important role in Ca 2+ influx. α3 and α4β2 as α-subunit sites of nAChR ion channels affect its activation. In our study, we found that SMI reduced CA synthesis and secretion induced by ACh, and cotreatment with SMI and the ACh receptor α-subunit inhibitors, HEX(α3) or DhβE(α4β2), did not produce any further inhibition, indicating that SMI may reduce CA synthesis and secretion by inhibiting α3 and α4β2 subunit sites to suppress AChR ion channels. As a rate-limiting enzyme in the synthesis of CA, the activity of TH is often tested. TH exerts its effect by phosphorylation at Ser19 with CaM kinase II and Ser40 with PKA. We found out that SMI suppressed ACh-induced TH activity by inhibiting the phosphorylation of TH at Ser19 and Ser40. KN-93 and H89, the antagonists of CaM kinase II and PKA, respectively, inhibited the ACh-induced phosphorylation at Ser19 and Ser40, and the addition of SMI did not augment the inhibitory effect. ese results indicate that SMI inhibits CA secretion by blocking TH activity at Ser19 and Ser40 sites. e present findings would support the idea that SMI suppresses the CA secretion and synthesis by inhibiting AChR ion channels and TH activity. SMI may also delay the development of cardiovascular disease through the modulation of the sympathetic-adrenal medullary system, primarily by inhibiting the secretion of neurotransmitters at the sympathetic terminals. To confirm this possibility, further in vivo and in vitro studies will be required in near future.