ABSTRACT AIM: To investigate the effect of magnesium sulfate and its interaction with the non-depolarizing muscle relaxant vecuronium at adult muscle-type acetylcholine receptors _in vitro_.

METHODS: Adult muscle-type acetylcholine receptors were expressed in HEK293 cells. Drug-containing solution was applied via a gravity-driven perfusion system. The inward currents were

activated by brief application of acetylcholine (ACh), and recorded using whole-cell voltage-clamp technique. RESULTS: Magnesium sulfate (1–100 mmol/L) inhibited the inward currents induced

ACh (10 μmol/L) in a concentration-dependent manner (IC50=29.2 mmol/L). The inhibition of magnesium sulfate was non-competitive. In contrast, vecuronium produced a potent inhibition on the

synergism between magnesium sulfate and non-depolarizing muscle relaxants at adult muscle-type acetylcholine receptors. SIMILAR CONTENT BEING VIEWED BY OTHERS STRUCTURAL INTERPLAY OF

ANESTHETICS AND PARALYTICS ON MUSCLE NICOTINIC RECEPTORS Article Open access 01 June 2023 STRUCTURAL MECHANISM OF MUSCLE NICOTINIC RECEPTOR DESENSITIZATION AND BLOCK BY CURARE Article 17

March 2022 CLC-1 INHIBITION AS A MECHANISM FOR ACCELERATING SKELETAL MUSCLE RECOVERY AFTER NEUROMUSCULAR BLOCK IN RATS Article Open access 28 October 2024 INTRODUCTION Magnesium sulfate

(MgSO4) has long been used in the treatment of eclampsia or placenta previa, which leads anesthesiologists to more frequent encounters with obstetric patients with hypermagnesemia. The

choice of anesthetic techniques for Cesarean sections in such patients is the subject of intense debate1, 2, 3. Although anesthesiologists prefer epidural anesthesia in hypermagnesemic

parturients, the risk of coagulopathy or hemodynamic catastrophe often requires anesthesiologists to proceed with urgent general anesthesia. However, some studies have shown that

hypermagnesemia increases the potency of non-depolarizing muscle relaxants, even magnesium, in the range of therapeutic serum concentrations4, 5, 6. Krendal7 suggested that magnesium sulfate

has a pre-synaptic effect by inhibiting acetylcholine release at motor nerve terminals, effects that may be responsible for the interaction with vecuronium. Furthermore, some studies have

shown that increasing extracellular magnesium reduces the size of acetylcholine-evoked responses and the single-channel conductance of nicotinic acetylcholine receptors at the mouse

end-plate8, 9, 10. However, no data regarding a combined effect of magnesium sulfate and non-depolarizing muscle relaxants on nicotinic acetylcholine receptors are available in the

literature. To evaluate the mechanism of inhibition of magnesium sulfate and non-depolarizing muscle relaxants on nicotinic acetylcholine receptors, we heterologously expressed nicotinic

acetylcholine receptors in human embryonic kidney 293 (HEK293) cells and studied the discrete and combined effects of magnesium sulfate with vecuronium, a non-depolarizing muscle relaxant

that is often used in general anesthesia. MATERIALS AND METHODS CELL CULTURE AND TRANSFECTION Expression plasmids [Psp65α, Psp65β, Psp65δ, and Pbssk(+)ɛ], encoding complementary DNA

sequences for the mouse muscle nicotinic acetylcholine receptor subunits (α, β, δ, and ɛ, respectively), were provided by the Salk Institute, USA. These coding sequences were subcloned into

pcDNA3.1+ (Invitrogen Life Technologies, Carlsbad, CA, USA). Human embryonic kidney 293 (HEK293) cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Grand

Island, NY, USA) supplemented with 10% calf serum (Invitrogen, Grand Island, NY, USA), 100 units/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C in a 5% CO2 incubator. The HEK293

cells were stably transfected with Lipofectamine 2000 according to the manufacturer's protocol (Invitrogen Life Technologies, Carlsbad, CA, USA). After transfection, the positive cell

clones were selected with G418, an aminoglycoside related to gentamicin, which is commonly used as a selective agent for eukaryotic cells. The transfected cells were then incubated for 24 h

before the recordings were made. ELECTROPHYSIOLOGY HEK293 cells were voltage-clamped using the whole-cell patch-clamp technique. Pipettes were pulled from borosilicate glass tubes with a

P-97 electrode puller (Sutter Instrument Co, Novato, CA, USA) at a resistance of 2–3 MΩ. The pipette electrode was filled with the following solution (mmol/L): CsCl, 140; MgCl2, 0.8;

HEPES-CsOH, 10; EGTA, 0.5; and Na-ATP, 4 (pH 7.3). The external solution contained the following (mmol/L): NaCl, 140; KCl, 2.5; CaCl2, 2; MgCl2, 0.8; HEPES-NaOH, 10; and glucose, 10 (pH

7.3). The MgSO4 solution was composed of the following (mmol/L): NaCl, 140; KCl, 2.5; MgSO4, 1, 10, 30, 50, and 100; HEPES-NaOH, 10; and glucose, 10 (pH 7.3). The cells were voltage-clamped

at −80 mV in the whole-cell configuration, and all of the experiments were performed at room temperature (20–24 °C). The currents were measured with an EPC10 amplifier (HEKA Elektronik,

Germany) and PatchMaster software (HEKA Elektronik, Germany), sampled at 20 kHz and stored on a computer. Acetylcholine and magnesium sulfate were purchased from Sigma (Sigma Chemical Co, St

Louis, MO, USA). Vecuronium (NV; Organon, the Netherlands) was obtained in preparations for clinical use, dissolved in the external solution or MgSO4 solution and applied by a

gravity-driven perfusion system. The solutions and dilutions for the experimental concentrations were prepared immediately before the experiments. The test solutions, containing either

acetylcholine alone or in combination with various concentrations of vecuronium or/and magnesium sulfate, were applied for 1 s to the HEK293 cells, and the peak current was then determined.

To evaluate the effect of the antagonist on the acetylcholine-elicited current, the solution containing vecuronium or/and magnesium sulfate was perfused on the HEK293 cells for 1 min prior

to the application of acetylcholine in the presence of the antagonist. The washout time between each drug application was at least 60 s to minimize the amount of desensitization throughout

the course of the experiment. Current was generated in five HEK293 cells. The control current in response to acetylcholine alone was repeated after the washout of the antagonist. Taking the

mean value of these two acetylcholine applications as the average control current, the antagonist response was calculated (percentage inhibition of average control current) using the

following equation: STATISTICAL ANALYSIS Data analysis was performed offline using Origin 8 (OriginLab, Northampton, MA, USA) and GraphPad Prism 4 (Graphpad Software, Inc, San Diego, CA,

USA). Concentration-response curves were fitted to the four-parameter logistic equation by non-linear regression analysis, and the IC50 values were determined. The results are expressed as

the mean±SD or as the 95% confidence interval (CI). Statistical significance was assessed with paired two-tailed Student's _t_-tests. A _P_<0.05 was considered statistically

significant. RESULTS Acetylcholine at different concentrations was applied for 1 s to HEK293 cells, which were voltage clamped at −80 mV and expressed the adult muscle-type acetylcholine

receptor. The acetylcholine induced inward currents in a dose-dependent fashion. The data were fitted to the logistic equation, and the acetylcholine concentrations producing 50% of the

maximal response (EC50) were 19.1 μmol/L (95% CI, 7.9–46.1; Figure 1). MAGNESIUM SULFATE INHIBITED ADULT MUSCLE-TYPE ACETYLCHOLINE RECEPTORS Magnesium sulfate produced a reversible,

concentration-dependent inhibition of 10 μmol/L acetylcholine-induced currents. Fitting the concentration responses of magnesium sulfate to the Hill equation yielded IC50 values of 29.2

mmol/L (95% CI, 14.7–58.0; Figure 2). The inhibitory effect of magnesium sulfate on the adult muscle-type acetylcholine receptors was then studied in more detail. The inward currents evoked

from the nicotinic acetylcholine receptors at different acetylcholine concentrations, in the absence and presence of magnesium sulfate, are shown in Figure 3. The peak inward currents under

control conditions were dependent on the acetylcholine concentration. Doses of 3 mmol/L and 30 mmol/L magnesium sulfate reduced the nicotinic acetylcholine receptor-mediated inward currents

at all of the acetylcholine concentrations studied. Increasing the concentrations of acetylcholine did not overcome the inhibition produced by magnesium sulfate. VECURONIUM INHIBITED ADULT

MUSCLE-TYPE ACETYLCHOLINE RECEPTOR Vecuronium also produced a potent inhibition of the adult muscle-type acetylcholine receptors. Fitting the concentration responses of vecuronium to the

Hill equation yielded IC50 values of 8.7 nmol/L (95% CI, 5.5–13.6; Figure 4). Furthermore, at increasing acetylcholine concentrations, there was a significant decrease in the inhibition of

the acetylcholine currents produced by 10 nmol/L vecuronium. At an agonist concentration of 1 μmol/L acetylcholine, 10 nmol/L vecuronium produced an inhibition of 91%±7% (_n_=5), whereas at

100 μmol/L acetylcholine, the inhibition was only 9%±2% (_n_=5), consistent with a competitive inhibition (Figure 5). INTERACTION BETWEEN MAGNESIUM SULFATE AND VECURONIUM ON ADULT

MUSCLE-TYPE ACETYLCHOLINE RECEPTORS To explore the interaction between magnesium sulfate and vecuronium on the function of the adult muscle-type acetylcholine receptor, we co-applied

magnesium sulfate and vecuronium at different concentrations to adult muscle-type acetylcholine receptors. The different concentrations of magnesium sulfate resulted in a strong enhancement

of the 10 nmol/L vecuronium-induced inhibition (_P_<0.01; Figure 6). Representative recordings of the raw data for both magnesium sulfate and vecuronium are shown in Figure 7. DISCUSSION

It has been shown that the action of non-depolarizing muscle relaxants is significantly prolonged in the presence of therapeutic serum levels of magnesium11. The mechanism of interaction for

this process was considered to be the pre-synaptic effects of magnesium sulfate; however, this study found that this potentiation of non-depolarizing muscle relaxants by magnesium can be

attributed in part to a combined effect on adult muscle-type acetylcholine receptors. Our study showed that magnesium sulfate produced a concentration-dependent inhibition of the adult

muscle-type acetylcholine receptor-mediated inward currents. The IC50 value of magnesium sulfate was 29.2 mmol/L, which far exceeds the serum magnesium concentrations in therapeutic ranges5,

6 and suggested that, in the range of therapeutic concentrations, magnesium alone could not attain a clinically acceptable neuromuscular inhibition. Our findings also showed that the

inhibition of the nicotinic acetylcholine receptor function by magnesium sulfate was insurmountable by merely increasing the concentrations of acetylcholine, indicating that magnesium

sulfate acts as a non-competitive inhibitor of the adult muscle-type acetylcholine receptor. This effect has been ascribed to the high-affinity interactions of divalent cations with negative

residues in the channel that decrease the local ion concentration12. Consistent with previous studies13, 14, 15, our study also showed that vecuronium potently inhibited the adult

muscle-type acetylcholine receptor. It is widely recognized that non-depolarizing muscle relaxants inhibit nicotinic acetylcholine receptors by a competitive mechanism16, 17. In this study,

an increasing acetylcholine concentration up to 500 μmol/L was able to overcome the inhibition of vecuronium on adult muscle-type acetylcholine receptors, confirming that vecuronium acts in

a competitive manner. To determine whether magnesium sulfate and vecuronium have an interactive effect, we observed the interaction between three different concentrations of magnesium

sulfate (1, 3, and 6 mmol/L) and vecuronium (10 nmol/L). Consistent with previous work4, 5, 6, our findings also showed a marked enhancement of the potency of vecuronium by magnesium sulfate

with all three of the concentrations tested. Fuchs-Buder _et al_6 reported that an intravenous infusion of magnesium at 40 mg/kg significantly potentiated the neuromuscular blockage of

vecuronium, and plasma magnesium concentrations increased from a baseline concentration of 0.9±0.06 to 1.08±0.07 mmol/L 15 min after the magnesium sulfate infusion (_P_<0.05). Although

this increase in plasma concentration of magnesium in patients pre-treated with magnesium sulfate was statistically significant, the administration of these doses was deemed clinically safe,

as no symptoms of muscle weakness were reported by the patients. In addition, Baraka and Yazigi18 found no clinical or electromyographic signs of muscle weakness, even at slightly higher

plasma magnesium concentrations (1.7–2.5 mmol/L). Thus, in the present study, the potentiation of vecuronium by a 1 mmol/L magnesium may have more clinical significance. During the course of

our study, we used a concentration of 10 μmol/L of acetylcholine as an agonist. This concentration can ensure sufficient acetylcholine current responses and minimize the desensitization of

the nicotinic acetylcholine receptor that resulted from the repetitive application of acetylcholine13, 14. In this study, the HEK293 cells were clamped at −80 mV because the antagonistic

effects of non-depolarizing muscle relaxants are independent of the holding voltages that range from −100 to −40 mV13, 15, 19, 20. CONCLUSION In conclusion, our results demonstrate that the

presence of magnesium in the range of therapeutic serum concentrations could significantly intensified the potency of vecuronium on adult muscle-type acetylcholine receptors. These results

suggest that the interaction between magnesium and non-depolarizing muscle relaxants on adult muscle-type acetylcholine receptors can partly explain how magnesium enhances a non-depolarizing

muscle relaxant-induced neuromuscular blockade. AUTHOR CONTRIBUTION Hong WANG and Shi-tong LI designed the research; Hong WANG and Qi-sheng LIANG performed the research; Xiao-hong LI and

Lan-ren CHENG analyzed the data; and Hong WANG, Wei FU, and Wen-tao DAI wrote the paper. ABBREVIATIONS MgSO4, magnesium sulfate; HEK293, human embryonic kidney 293; nAChR, nicotinic

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references ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (No 30571796). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of

Anesthesiology, the First Affiliated Hospital of Bengbu Medical College, Bengbu, 233000, China Hong Wang, Qi-sheng Liang, Lan-ren Cheng, Xiao-hong Li, Wei Fu & Wen-tao Dai * Department

of Anesthesiology, First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200030, China Hong Wang & Shi-tong Li Authors * Hong Wang View author

publications You can also search for this author inPubMed Google Scholar * Qi-sheng Liang View author publications You can also search for this author inPubMed Google Scholar * Lan-ren Cheng

View author publications You can also search for this author inPubMed Google Scholar * Xiao-hong Li View author publications You can also search for this author inPubMed Google Scholar *

Wei Fu View author publications You can also search for this author inPubMed Google Scholar * Wen-tao Dai View author publications You can also search for this author inPubMed Google Scholar

* Shi-tong Li View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Shi-tong Li. RIGHTS AND PERMISSIONS Reprints and

permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Wang, H., Liang, Qs., Cheng, Lr. _et al._ Magnesium sulfate enhances non-depolarizing muscle relaxant vecuronium action at adult muscle-type

nicotinic acetylcholine receptor _in vitro_. _Acta Pharmacol Sin_ 32, 1454–1459 (2011). https://doi.org/10.1038/aps.2011.117 Download citation * Received: 16 June 2011 * Accepted: 20 July

2011 * Published: 10 October 2011 * Issue Date: December 2011 * DOI: https://doi.org/10.1038/aps.2011.117 SHARE THIS ARTICLE Anyone you share the following link with will be able to read

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KEYWORDS * magnesium sulfate * non-depolarizing muscle relaxant * vecuronium * adult muscle-type acetylcholine receptor * synergism