Different Ca²⁺ releasing action of caffeine and depolarisation in skeletal muscle fibres of the rat

Abstract

The relative abilities of caffeine and transverse tubular (T‐) system depolarisation to induce Ca2+ release in mammalian skeletal muscle were compared in mechanically skinned fibres of the rat, in order to determine whether normal excitation‐contraction (E‐C) coupling is achieved by up‐regulating the Ca2+‐induced Ca2+ release process, as caffeine is known to do. Caffeine triggered Ca2+ release in soleus (slow‐twitch) fibres at much lower concentrations than in extensor digitorum longus (EDL) (fast‐twitch) fibres when the sarcoplasmic reticulum (SR) of each type was loaded with Ca2+ at close to endogenous levels. The difference in caffeine sensitivity resulted at least in part from the SR being loaded endogenously at near maximal capacity in soleus fibres but at less than half of maximal capacity in EDL fibres. The caffeine sensitivity could be reversed by reversing the relative level of SR loading. The ability of caffeine to induce Ca2+ release was markedly reduced by lowering the level of SR loading or by raising the free [Mg2+] from 1 to 3 mm. Caffeine, even at 30 mm, triggered little or no Ca2+ release in EDL fibres (a) at 1 mm (physiological) Mg2+ when the SR was loaded at two‐thirds or less of the endogenous level, and (b) at 3 mm Mg2+ when the SR was loaded at close to the endogenous level. In contrast, depolarisation potently elicited Ca2+ release under these conditions in the same fibres. The inability of 30 mm caffeine to induce Ca2+ release under certain conditions was not attributable to desensitisation or inactivation of the release channels, because there was no response even upon initial exposure to caffeine and depolarisation always remained able to trigger Ca2+ release. It instead appeared that caffeine was a relatively ineffectual stimulus in EDL fibres except under conditions where (a) the SR was heavily loaded, (b) the free [Mg2+] was low, or (c) a high [Cl−] was present. These results show that the normal E‐C coupling mechanism in mammalian skeletal muscle does not involve just enhancing Ca2+‐induced Ca2+ release, and evidently requires the removal or bypassing of the inhibitory effect of Mg2+ on the Ca2+ release channels.

Functional implications of modifying RyR‐activating peptides for membrane permeability

“…Skeletal SR (100 mg of protein) was added to the cuvette solution (final volume of 2 ml), containing (mM): KH 2 PO 4 , 100 (pH ¼ 7); MgCl 2 , 4; Na 2 ATP, 1; antipyrylazo III, 0.5). Ca 2 þ , Mg 2 þ -ATPase activity was suppressed with thapsigargin (200 nM; Sagara & Inesi, 1991 (Lamb et al, 2001). To test the sensitivity to 8 mM caffeine (e.g.…”

“…To test the sensitivity to 8 mM caffeine (e.g. Figure 9), the SR was loaded with Ca 2 þ to a set level (close to that in the fibre endogenously) by exposing the fibre for a set period (usually 15 s) to a solution buffered at pCa 6.7 with 1.0 mM total EGTA (Lamb et al, 2001). The fibre was then equilibrated in standard weakly Ca 2 þ -buffered solution (1 mM Mg 2 þ , 0.05 mM EGTA, pCa 7.1) for 20 s, with or without peptide, and exposed to a similar solution with 8 mM caffeine for 15 s, before fully depleting the SR of its remaining Ca 2 þ with the Full Release Solution.…”

Congenital muscle disorders with cores: the ryanodine receptor calcium channel paradigm

“…To discriminate between these two pathogenetic hypotheses it is crucial to define (i) SR Ca 2+ content in muscle cells from CCD patients carrying mutation in the COOH-terminal domain, and (ii) clearly establish the role of SR Ca 2+ load in the regulation of Ca 2+ release in mammalian fibres [39]. If the Ca 2+ leak hypothesis is correct, the prevalent involvement of type I fibres suggests that the extent of Ca 2+ leak via mutated RyR is higher in slow fibres compared to fast fibres, and likely reflects larger SR Ca 2+ load of slow twitch muscles [40]. A knock-in CCD mouse model carrying the heterozygous RyR1 I4895T mutation does not reconstitute the severe CCD skeletal muscle phenotype of humans harbouring homologous heterozygous RYR1 mutations [41].…”

Fast kinetics of calcium dissociation from calsequestrin

“…Results obtained in skinned skeletal muscle cells, which show that Ca 2+ release rates depend on Ca 2+ loading (Lamb et al, 2001), also support a role of CSQ in regulating RyR activity. In intact cardiomyocytes, increased or decreased cardiac CSQ expression is an important determinant of the Ca 2+ storage capacity of the SR (Gyorke et al, 2004).…”