A study of the short-term effects of glucose 6-phosphate on the contractile activation properties of skinned single muscle fibres of the rat. Implications for solution design

Abstract.

The present study highlights possible problems that can arise from the incorrect preparation of control and test solutions for use in Ca²⁺-activation experiments using single skinned skeletal muscle fibres and EGTA-based Ca²⁺ buffers. We show here, using glucose 6-phosphate (G6-P) as our "test" compound, that the Ca²⁺-activation properties of skinned single fibre segments from the extensor digitorum longus muscle of the rat are highly dependent on the form in which the G6-P is added and on the correct balancing of an appropriate anion in control solutions. Test solutions prepared by the direct addition of 10 mM monosodium G6-P salt to a set of control solutions of defined pCa resulted in significantly greater submaximal force responses than the corresponding controls. This is equivalent to an increase in the sensitivity of the contractile-regulatory system to Ca²⁺ (pCa₅₀=–log₁₀[Ca²⁺] that produces 50% of maximum force) by 0.19±0.01 pCa units. In contrast, addition of disodium G6-P to control solutions caused a slight reduction in the apparent sensitivity of the contractile apparatus to Ca²⁺ by 0.04±0.01 pCa units (P<0.01). Rather than being indicative of the effects of G6-P on the contractile apparatus, these opposing effects are due to differences between test and control solutions with respect to pH and Na⁺ concentration brought about by the G6-P salts. When all ionic species were carefully balanced, 10 mM G6-P was found to have only a small sensitizing effect on Ca²⁺-activation properties compared to control, without affecting the maximum Ca²⁺-activated force response. Our findings highlight the often-overlooked need for careful balancing of the ionic composition in control and test solutions when examining the true effects of different compounds on the Ca²⁺-activation characteristics of single skinned muscle fibre preparations.