Effect of Hydrogen sulphide (H2S) on the electrophysiological properties of bovine articular chondrocytes

Vasanthakumar, N (2018) Effect of Hydrogen sulphide (H2S) on the electrophysiological properties of bovine articular chondrocytes. Masters thesis, Christian Medical College, Vellore.

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Abstract

INTRODUCTION: It is well known that gaseotransmitters such as hydrogen sulphide (H2S) and nitric oxide (NO) play a key role in bone-joint pathophysiology. The effect of H2S and NO on the chondrocyte membrane potential and its effect on bovine chondrocytes ion currents have not been studied so far. OBJECTIVES: 1. To study the effect of H2S on potassium currents in bovine chondrocytes using the voltage clamp technique. 2. To study the effect of L-Arginine and lipopolysaccharide (LPS) on potassium currents in bovine articular chondrocytes using the voltage clamp technique. 3. To study the effect of H2S, L-Arginine, N-ω-Nitro-L-Arginine (LNNA) and LPS on the membrane potential of bovine articular chondrocytes using the current clamp technique. MATERIALS AND METHODS: Patch clamp recordings were performed using the Axopatch 200B amplifier in the whole cell configuration. Membrane potential and potassium currents of bovine chondrocytes (freshly isolated and cultured) were recorded in the current clamp mode and the voltage clamp mode respectively. Tetraethylammonium (TEA, a potassium channel blocker), L-Arginine (a substrate for nitric oxide synthases), sodium hydrosulfide (NaHS, a H2S donor) and N-ω-nitro-L-arginine (LNNA, a nitric oxide synthase inhibitor) were used to assess their effect on the membrane potential and potassium currents. Lipopolysaccharide (LPS) was used to mimic inflammatory condition. Standard extracellular and pipette solutions were used. RESULTS: Results were expressed as mean ± SEM. The membrane capacitance (Cm) of the bovine articular chondrocytes was 7.01± 0.38pF (n=84). The resting membrane potential of bovine articular chondrocytes was -24.26±3.63 mV (n=14). The chondrocyte membrane potential, with the addition of 500uM NaHS changed from -31.10±7.52 to -29.19±8.36mV (P=0.6, n=6), with 2mM NaHS from -33.55±6.83 to -30.43±7.80mV (P=0.173, n=6), with 1 mM L-Arginine from -23.98±6.43 to -27.83±5.01mV (P= 0.374, n=9), with 10mM TEA from -24.99±3.67 to -20.21±3.26mV (P=0.221, n=14), with 2mM LNNA from -30.03±9.80 to 0.53±1.86mV (P=0.109, n=3). Voltage clamp studies at 60 mV showed that the mean peak current density on addition of 500uM NaHS changed from 370.68±115.71 pA/pF to 411.93 ± 117.92 pA/pF (P= 0.3, n=14), with 2mM NaHS from 162.53 ± 100.83 pA/pF to 306.54 ± 148.42 pA/pF (P=0.144, n=4), with 8μg/ml LPS on the first exposure from 729.60 ± 248.20 pA/pF to 688.73 ± 239.05 pA/pF (P=0.465, n=4), with 8μg/ml LPS on the second exposure from 535.45 ± 218.69 pA/pF to 434.34 ± 103.34 pA/pF (P=0.593, n=3), with 10mM TEA from 249.53 ± 54.23 pA/pF to 139.93 ± 47.37 pA/pF (P=0.091, n=7). A tail current protocol showed a reversal potential (Erev) of - 60mV. CONCLUSION: TEA results suggest that the currents seen in the bovine chondrocytes are due to the opening of voltage gated delayed rectifier potassium channels. A depolarization of membrane potential with TEA indicates that potassium channels may play a role in the maintenance of the chondrocyte membrane potential. A depolarization of the membrane with LNNA suggests that Nitric oxide may also play a role in chondrocyte membrane potential regulation. Although the membrane potential and current density changes produced by NaHS, L-arginine and LPS were statistically insignificant, the trend observed suggests the need to further study this phenomenon with a larger sample size.

Item Type: Thesis (Masters)
Uncontrolled Keywords: Bovine chondrocytes ; membrane potential ; hydrogen sulphide ; nitric oxide ; LNNA ; L-Arginine ; Lipopolysaccharide.
Subjects: MEDICAL > Physiology
Depositing User: Subramani R
Date Deposited: 17 Aug 2018 03:01
Last Modified: 17 Aug 2018 03:01
URI: http://repository-tnmgrmu.ac.in/id/eprint/9284

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