Korean J Physiol Pharmacol.  2011 Oct;15(5):285-289. 10.4196/kjpp.2011.15.5.285.

Glycine- and GABA-mimetic Actions of Shilajit on the Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis in Mice

• Yin H
• Yang EJ
• Park SJ
• Han SK

• KMID: 2071731
• DOI: http://doi.org/10.4196/kjpp.2011.15.5.285

Abstract

Shilajit, a medicine herb commonly used in Ayurveda, has been reported to contain at least 85 minerals in ionic form that act on a variety of chemical, biological, and physical stressors. The substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc) are involved in orofacial nociceptive processing. Shilajit has been reported to be an injury and muscular pain reliever but there have been few functional studies of the effect of Shilajit on the SG neurons of the Vc. Therefore, whole cell and gramicidin-perfotrated patch clamp studies were performed to examine the action mechanism of Shilajit on the SG neurons of Vc from mouse brainstem slices. In the whole cell patch clamp mode, Shilajit induced short-lived and repeatable inward currents under the condition of a high chloride pipette solution on all the SG neurons tested. The Shilajit-induced inward currents were concentration dependent and maintained in the presence of tetrodotoxin (TTX), a voltage gated Na+ channel blocker, CNQX, a non-NMDA glutamate receptor antagonist, and AP5, an NMDA receptor antagonist. The Shilajit-induced responses were partially suppressed by picrotoxin, a GABAA receptor antagonist, and totally blocked in the presence of strychnine, a glycine receptor antagonist, however not affected by mecamylamine hydrochloride (MCH), a nicotinic acetylcholine receptor antagonist. Under the potassium gluconate pipette solution at holding potential 0 mV, Shilajit induced repeatable outward current. These results show that Shilajit has inhibitory effects on the SG neurons of Vc through chloride ion channels by activation of the glycine receptor and GABAA receptor, indicating that Shilajit contains sedating ingredients for the central nervous system. These results also suggest that Shilajit may be a potential target for modulating orofacial pain processing.

Keyword

Substantia gelatinosa neurons; Shilajit; Patch clamp; Glycine receptor; GABAA receptor

MeSH Terms

6-Cyano-7-nitroquinoxaline-2,3-dione
Animals
Brain Stem
Central Nervous System
Chloride Channels
Facial Pain
Gluconates
Mecamylamine
Mice
Minerals
N-Methylaspartate
Neurons
Picrotoxin
Potassium
Receptors, Glutamate
Receptors, Glycine
Receptors, Nicotinic
Resins, Plant
Strychnine
Substantia Gelatinosa
Tetrodotoxin
6-Cyano-7-nitroquinoxaline-2,3-dione
Chloride Channels
Gluconates
Mecamylamine
Minerals
N-Methylaspartate
Picrotoxin
Potassium
Receptors, Glutamate
Receptors, Glycine
Receptors, Nicotinic
Resins, Plant
Strychnine
Tetrodotoxin

Figure

• 

  Fig. 1. Shilajit-induced currents are repeatable and mediated by postsynaptic cell actions on SG neurons. (A) A representative trace showing the repeatable inward currents induced by Shilajit (300μ g/ml). (B) A representative trace showing the inward current induced by Shilajit (300μg/ml) alone and in the presence of TTX (0.5μM). (C) Relative bars showing a comparison of the membrane current changes by the repeated application of Shilajit (300μg/ml, n=8, paired t-test). (D) Relative bars showing a comparison of the membrane current changes by the Shilajit alone and in the presence of TTX (300μg/ml, n=5, paired t-test).

• 

  Fig. 2. Shilajit-induced concentration dependent responses on SG neurons. Curve figure showing the response of 30, 100, 300μg/ml and 1, 3 mg/ml Shilajit (n=8). EC50 was estimated to 562μg/ml.

• 

  Fig. 3. Shilajit-activated currents were not mediated by the NMDA and non-NMDA glutamate receptors on the SG neurons. (A, B) Representative traces showing the inward current induced by Shilajit application (300μg/ml). The Shilajit induced inward current persisted in the presence AP5 (NMDA receptor antagonist) and CNQX (non-NMDA receptor antagonist). (C, D) Relative responses of Shilajit in the presence of AP5 or CNQX compared to Shilajit alone (n=5).

• 

  Fig. 4. Silajit-induced inward currents are mediated by the activation of glycine and GABAA receptors. (A) Representative trace showing the inward current induced by Shilajit (300μg/ml). Shilajit-induced inward current was reduced in the presence of picrotoxin (PIC, GABAA receptor antagonist). (B) A representative trace showing the inward current induced by Shilajit application (300μg/ml) in whole-cell recording. The Shilajit-induced inward current was reduced in the presence of strychnine. (C) Relative response of Shilajit in the presence of PIC compared to Shilajit alone (n=4), ∗p<0.05. (D) Membrane current change in Shilajit alone and in the presence of strychnine (STR, a glycine receptor antagonist, n=4) ∗p<0.05. (E) A representative trace showing hyperpolarization induced by Shilajit application (1 mg/ml) in gramicidin-perforated recording (RMP=–54 mV). Shilajit-induced hyperpoalrization was blocked in the presence of strychnine. Membrane potential change in Shilajit alone and in the presence of strychnine (STR, n=3) ∗p<0.05. (F) A representative trace showing the repeatable outward currents induced by Shilajit (300μg/ml) under potassium gluconate pipette solution at VH=0 mV.

• 

  Fig. 5. Silajit-induced inward currents are not mediated by the activation of nACh receptors. (A) Representative trace showing the inward current induced by Shilajit (300μg/ml). Shilajit-induced inward current was not affected in the presence of MCH (nACh receptor antagonist). (B) Relative response of Shilajit in the presence of MCH compared to Shilajit alone (n=4), ∗p<0.05.

Reference

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