Effect of muscle contractions on interstitial ATP of perfused muscle with or without inhibitors.

<p>Blood-perfused rat gastrocnemius muscle was subjected to repeated muscle contractions at 1 Hz in the absence of inhibitors (A) or in the presence of CFTR_inh-172 (20 µM; B), KT5720 (10 µM; C), amiloride (100 µM; D) or SN-6 (20 µM; E). Values are the mean ± SEM of 6 (A), 11 (B), 7 (C), 8 (D) or 5 (E) tests. *, significant difference between rest and contractions; #, significant difference between absence and presence of inhibitor (1-way repeated measures ANOVA followed by the Bonferroni post-hoc t-test).</p

Influence of lactic acid on the induction of pCREB and influence of forskolin on CFTR phosphorylation in L6 cultured myocytes.

<p>A: sample gel showing the bands for pCREB, CREB and β-actin, which served as the internal control. Band density for pCREB (B) or total CREB (C) was expressed as a ratio to β-actin. Values are the mean ± SEM of 3 tests. *, significantly different from the value in the absence of lactic acid in 1-way ANOVA followed by the Bonferroni post-hoc t-test. D: sample autoradiograph showing the change in CFTR phosphorylation in response to forskolin (20 µM), forskolin with KT5720, or forskolin plus dibutyryl-cAMP (200 µM) and IBMX (1 mM). E: densitometry analysis of CFTR phosphorylation under the same conditions.</p

Influence of lactic acid on the extracellular and intracellular ATP of L6 cultured myocytes.

<p>A: time-course for the appearance of ATP in the bathing medium during exposure to 10 mM lactic acid. Values are the mean ± SEM of 14–30 values; *, significantly different from its own time-control in Student's t-test. B: appearance of ATP in the bathing medium following 3 hrs exposure to 10 mM lactic acid in the absence of inhibitors or in the presence of CFTR_inh-172 (20 µM) or glibenclamide (200 µM). Values are the mean ± SEM of 9–12 tests. *, significant difference between the presence and absence of lactic acid in Student's t-test; #, significant difference between the presence or absence of the inhibitor in 1-way ANOVA followed by the Bonferroni post-hoc t-test. C: effect of incubation for 30 mins in different concentrations of lactic acid on the intracellular ATP. Values are the mean ± SEM of 6–12 values; *, significantly different from 0 mM lactic acid in 1-way ANOVA followed by the Bonferroni post-hoc test.</p

Influence of lactic acid on interstitial ATP of buffer-perfused soleus muscle of anaesthetised rats.

<p>Effect of lactic acid on interstitial ATP in the absence of inhibitors (A) or in the presence of α-cyano-4-hydroxycinnamic acid (2 mM; B), CFTR_inh-172 (20 µM; C) or glibenclamide (200 µM; D). Values are the mean ± SEM of 14–17 values in A, 6 values in B, 7 values in C or 6 values in D. *, significantly different from the pre-control in 1-way repeated measures ANOVA followed by the Bonferroni post-hoc t-test.</p

Involvement of PKA in lactic-acid-induced ATP release from cultured myocytes or perfused muscle.

<p>A: PKA activity was measured in the supernatant from the L6 myocyte lysate following 30 mins incubation of the cells with the concentrations of lactic acid shown. The upper sample gel shows the separated bands of phosphorylated and non-phosphorylated peptide, and the lower gel shows the internal standard, β-actin, which was measured to confirm that the loading of the gel was kept constant. B: Appearance of ATP in the bathing medium of L6 myocytes exposed to 10 mM lactic acid in the absence or presence of KT5720 for 3 hrs. Values are the mean ± SEM of 9–13 tests. *, significant difference between the absence or presence of lactic acid; #, significant difference between the absence or presence of KT5720 (1-way ANOVA followed by the Bonferroni post-hoc t-test). C & D: Appearance of ATP in the interstitial space of buffer-perfused rat soleus muscle during infusion of lactic acid (5 mM) in the absence (C) or presence (D) of KT5720. Values are the mean ± SEM of 11 (C) or 6 (D) tests. *, significant difference between the absence or presence of lactic acid in 1-way repeated measures ANOVA followed by the Bonferroni post-hoc t-test.</p

CFTR and AC isoform expression in L6 myocytes.

<p>A: sample gel showing RT-PCR of CFTR and the internal standard, β-actin, from cells incubated for 30 mins with the concentrations of lactic acid shown. C. CFTR mRNA expression was normalised as the ratio to β-actin. Values are the mean ± SEM of 4 tests. *, significant difference between the absence or presence of lactic acid in 1-way ANOVA followed by the Bonferroni post-hoc t-test. B: sample gel showing western blot of CFTR protein from cells incubated for 3 hrs in the absence or presence of 10 mM lactic acid. D: CFTR protein expression was normalised as the ratio to β-actin; both the mature (168 kDa) and immature (150 kDa) bands were quantitated. Values are the mean ± SEM of 8–9 tests. *, significant difference between the absence or presence of lactic acid in 1-way ANOVA followed by the Bonferroni post-hoc t-test. E: sample gel showing RT-PCR determination of AC isoforms expressed by L6 myocytes. F: AC mRNA expression was normalised as a ratio to GAPDH. Values are the mean ± SEM of 3 values.</p

Comparison between force generated in the first and second contractions of perfused muscle.

<p>Blood-perfused rat gastrocnemius muscle was subjected to repeated muscle contractions at 1 Hz in the absence of inhibitors (A) or in the presence of CFTR_inh-172 (20 µM; B), KT5720 (10 µM; C), amiloride (100 µM; D) or SN-6 (20 µM; E). Values are the mean ± SEM of 5–11 tests. There were no significant differences between the first and second contractions in the absence or presence of any inhibitors.</p

Attenuation of acid-induced increases in intracellular cAMP and extracellular ATP by SN6 or KB-R7943.

<p>A: L6 myocytes were incubated in the absence or presence of lactic acid (10 mM) for 30 mins, and values are the mean ± SEM of 6–9 tests. B: myocytes were incubated in the absence or presence of lactic acid (10 mM) for 3 hrs, and values are the mean ± SEM of 12–27 values. *, significant difference between the absence or presence of lactic acid; #, significant difference between the absence or presence of inhibitor (1-way ANOVA followed by the Bonferroni post-hoc t-test).</p

Role of intracellular cAMP in stimulating the increase in extracellular ATP at low pH.

<p>A: influence of forskolin (20 µM) on the interstitial ATP of buffer-perfused rat soleus muscle. Values are the mean ± SEM of 5 observations. *, significantly greater than the precontrol in 1-way repeated measures ANOVA followed by the Bonferroni post-hoc t-test. B: comparison of the effect of forskolin (20 µM) or lactic acid (10 mM) in the absence or presence of IBMX on the appearance of ATP in the bathing medium of L6 cultured myocytes. Values are the mean ± SEM of 12–27 tests. *, significantly different from its own control group in Student's t-test; #, significant difference between the absence or presence of IBMX (Student's t-test). C: influence of lactic acid or sodium lactate on the intracellular cAMP of L6 cultured myocytes. Values are the mean ± SEM of 15 values for control and of 6–9 values for treatments. *, significantly different from control in 1-way ANOVA followed by the Bonferroni post-hoc t-test. D: determination of the maximal cAMP response to forskolin in the L6 myocytes: the line shows the 5-parameter sigmoid curve fit (r = 1.0) which predicts a maximal cAMP response of 9.0 nM.</p

Influence of amiloride on lactic-acid-induced increases in myocytes intracellular cAMP and extracellular ATP.

<p>Influence of amiloride on the intracellular cAMP (A) and the extracellular ATP (B) of L6 cultured myocytes incubated for 30 mins in the absence or presence of lactic acid (10 mM). Values are the mean ± SEM of 6–9 tests (A) or 12–27 tests (B). *, significant difference between the absence or presence of lactic acid; #, significant difference between the absence or presence of amiloride (1-way ANOVA followed by the Bonferroni post-hoc t-test).</p