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

Abstract

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.

Figure 1

The effects of SMI on CA secretion in bovine adrenal medullary cells. (a) Effects of 2.5 μL/mL SMI on basal CA secretion and on various secretagogue agonists induced CA secretion. (b) Effects of SMI on CA secretion induced by 300 μM. Data are expressed in mean ± SEM based on three independent experiments each performed in triplicates (n = 6 per group). ^∗∗p < 0.01 compared with ACh alone. ^##p < 0.01 compared with untreated control.

Figure 2

Effects of combinations of SMI and several inhibitors of α-subunit sites in the nAChR ion channels on catecholamine secretion induced by 300 μM ACh. Data are expressed as mean ± SEM based on three independent experiments each performed in triplicates (n = 6 per group). ^##p < 0.01, compared with 300 μM ACh alone and ^∗∗p < 0.01, compared with 300 μM ACh plus SMI.

Figure 3

Effects of different concentrations of SMI on ACh-induced Ca²⁺ influx in cultured bovine adrenal medullary cells. (a) The cells were observed by an inverted fluorescent microscope at 200x amplified. (b) The fluorescence intensity was by detected by a microplate reader. Data are expressed as mean ± SEM based on three independent experiments each performed in triplicates (n = 6 per group). ^∗p < 0.05, ^∗∗p < 0.01 compared with ACh alone. ^##p < 0.01 compared with control.

Figure 4

Effect of various concentrations of SMI on ACh-induced activity of TH. Data are expressed as mean ± SEM based on three independent experiments each performed in triplicates (n = 6 per group). ^∗∗p < 0.01 compared with ACh alone. ^##p < 0.01 compared with control.

Figure 5

Effect of SMI on TH phosphorylation in bovine adrenal medullary cells. Phosphorylation at the Ser19, Ser31, and Ser40 sites of TH was determined and expressed as % of control TH band. (a) Effect of SMI on TH phosphorylation at Ser19, Ser31, and Ser40. (b) Effect of SMI on the ratio of the density of TH Ser19 phosphorylation induced by 300 μM ACh. (c) Effect of SMI on the ratio of the density of TH Ser31 phosphorylation induced by 300 μM ACh. (d) Effect of SMI on the ratio of the density of TH Ser40 phosphorylation induced by 300 μM ACh. ((e), (g)) Effect of SMI and CaM kinase II on phosphorylation of TH Ser19 induced by 300 μM ACh. ((f), (h)) Effect of SMI and PKA on phosphorylation of TH Ser40 induced by 300 μM ACh. Data are expressed as mean ± SEM based on three independent experiments each performed in triplicates. ^∗p < 0.05 and ^∗∗p < 0.01 compared with ACh alone. ^#p < 0.05 and ^##p < 0.01 compared with control.