Low pH up‐regulates interleukin‐6 mRNA in L6‐G8C5 rat skeletal muscle cells independent of pH sensing by SNAT2(SLC38A2) transporters

Abstract

Exercise is known to create a transient, but potent increase in skeletal muscle expression of potentially anti‐inflammatory myokine interleukin‐6 (IL‐6). This effect may be clinically important in managing chronic inflammatory states. It has previously been proposed that lactic acidosis following exercise promotes this IL‐6 up‐regulation, but the mechanism of this acidosis effect is unknown. Rat skeletal muscle cell line L6‐G8C5 has been used previously to model metabolic effects of acidosis, sensing low pH through the resulting inhibition of amino acid transporter SNAT2(SLC38A2). Use of ionophore ionomycin to model the rise in intracellular Ca2+ concentration occurring in contracting muscle strongly up‐regulates IL‐6 mRNA in L6‐G8C5 myotubes. This study used this model to test the hypothesis that low extracellular pH (7.1) enhances ionomycin‐induced IL‐6 mRNA up‐regulation by inhibiting SNAT2. Incubation of L6‐G8C5 myotubes for 6 h with 0.5 µM ionomycin at control pH (7.4) resulted in a 15‐fold increase in IL‐6 mRNA which was further enhanced (1.74‐fold) at pH 7.1. In contrast low pH had no significant effect on IL‐6 mRNA without ionomycin, nor on the IL‐6 mRNA increase that was induced by cyclic stretch. Even though pH 7.1 halved the transport activity of SNAT2, alternative methods of SNAT2 inhibition (JNK inhibitor SP600125; SNAT2 antagonist MeAIB; or SNAT2 silencing with siRNA) did not mimic the enhancing effect of low pH on IL‐6 mRNA. On the contrary, JNK inhibition blunted the effect of pH 7.1 with ionomycin, but had no effect at pH 7.4. It is concluded that low pH promotes Ca2+/ionomycin–induced up‐regulation of IL‐6 mRNA through a novel SNAT2‐independent JNK‐dependent pH‐sensing pathway not previously described in this skeletal muscle model.