Effects of terbutaline on force and intracellular calcium in slow-twitch skeletal muscle fibres of the rat

Abstract

1. The effect of the alpha2-adrenoceptor agonist, terbutaline, was investigated on simultaneously measured force and intracellular free calcium ([Ca2+]i) in intact rat soleus muscle fibres, and on contractile protein function and Ca2+ content of the sarcoplasmic reticulum (SR) in skinned fibres. 2. Terbutaline (10 microM) had no significant effect on either resting force or [Ca2+]i. Exposure to terbutaline increased both the integral of the indo-1 ratio transient and peak twitch force by 37%. 3. At sub-maximal (10 Hz) stimulation frequencies, terbutaline accelerated force relaxation but had highly variable effects on tetanic force amplitude. The corresponding indo-1 ratio transients were significantly larger, and faster to decay than the controls. 4. Terbutaline increased tetanic force at near maximal stimulation frequencies (50 Hz) by increasing tetanic [Ca2+]i. Force relaxation was accelerated at this frequency with no significant change in the indo-1 ratio transient decay rate. 5. All of terbutaline's effects on force and indo-1 ratio transients in intact fibres were completely blocked and reversed by ICI 118551 (1 microM). 6. Mechanically skinned fibres isolated from intact muscles pre-treated with terbutaline showed no significant changes in SR Ca2+ content, myofilament [Ca2+]i-sensitivity or maximum force generating capacity. 7. The results suggest that terbutaline primarily modulates force by altering the amplitude and decay rate of the [Ca2+]i transient via phosphorylation of both the ryanodine receptor (RR) and the SR pump regulatory protein, phospholamban (PLB). The high variability of responses of slow-twitch muscles to beta2-agonists probably reflects individual differences in basal phosphorylation levels of PLB relative to that of RR.

Figure 1

Effect of 10 μM terbutaline on force (upper traces) and indo-1 ratio transients (lower traces) recorded during twitches. (A and C) controls. (B and D) 30 min after terbutaline (10 μM) application. Traces are averages of 20 successive twitches elicited at 0.1 Hz (ratio traces filtered digitally at 100 Hz).

Figure 2

Effect of 10 μM terbutaline on force (upper traces) and indo-1 ratio transients (lower traces) recorded during sub-maximal (10 Hz) tetanic stimulation. (A and C) controls. (B and D) 20–40 min after terbutaline application. Traces are averages of four successive 10 Hz tetani given at 5 min intervals (ratio traces filtered digitally at 10 Hz).

Figure 3

Effect of 10 μM terbutaline on force (upper traces) and indo-1 ratio transients (lower traces) recorded during near maximal (50 Hz) tetanic stimulation. (A and C) controls. (B and D) 20–40 min after terbutaline (10 μM) application. Traces are averages of four successive 50 Hz tetani given at 5 min intervals (ratio traces filtered digitally at 10 Hz).

Figure 4

Comparison of the effects of 10 μM terbutaline on Ca²⁺-sensitivity of the contractile proteins in intact (A) and mechanically skinned (B) soleus muscle fibres. Results in (A) are normalized to the same maximum force (1.00). Calculated pCa₅₀ values (±95% confidence limits) were: control, 6.09±0.02; terbutaline, 6.03±0.01; wash, 6.07±0.005. (B) shows mean results (n=4–7) from mechanically-skinned fibres that were exposed to terbutaline whilst still intact. Curves represent the least squares computer-generated fit to a modified version of the Hill equation. Calculated values (±s.e.mean) for pCa₅₀: control, 6.11±0.02; terbutaline, 6.09±0.02.

Figure 5

Terbutaline, like caffeine, enhances the apparent maximum tetanic force by potentiating steady-state tetanic [Ca²⁺]_i. Points comprising the force-pCa relation were obtained from an indo-1 injected soleus muscle bundle in the following sequence: control, 15 min after 1 mM caffeine, 20 min after 1 mM caffeine plus 10 μM terbutaline. Note that the dotted line (indicating the apparent maximum Ca²⁺-activated force (P_o) under control conditions, ∼20 N.cm⁻²) is below the value attainable in either caffeine alone or caffeine plus terbutaline. A single curve was fitted to the combination of the three data sets yielding the following parameters (±95% confidence level): pCa₅₀, 6.30±0.05, P_o, 23.9±2.4 N.cm⁻².