Regulation of a voltage-sensitive release mechanism by Ca2+-calmodulin-dependent kinase in cardiac myocytes

A role for Ca2+-calmodulin-dependent kinase (CamK) in regulation of the voltage-sensitive release mechanism (VSRM) was investigated in guinea pig ventricular myocytes. Voltage clamp was used to separate the VSRM from Ca2+-induced Ca2+release (CICR). VSRM contractions and Ca2+ transients were absent in cells dialyzed with standard pipette solution but present when 2–5 μM calmodulin was included. Effects of calmodulin were blocked by KN-62 (CamK inhibitor), but not H-89, a protein kinase A (PKA) inhibitor. Ca2+ current and caffeine contractures were not affected by calmodulin. Transient-voltage relations were bell-shaped without calmodulin, but they were sigmoidal and typical of the VSRM with calmodulin. Contractions with calmodulin exhibited inactivation typical of the VSRM. These contractions were inhibited by rapid application of 200 μM of tetracaine, but not 100 μM of Cd2+, whereas CICR was inhibited by Cd2+ but not tetracaine. In undialyzed myocytes (high-resistance microelectrodes), KN-62 or H-89 each reduced amplitudes of VSRM contractions by ∼50%, but together they decreased VSRM contractions by 93%. Thus VSRM is facilitated by CamK or PKA, and both pathways regulate the VSRM in undialyzed cells.

GTP gammaS removal of D-600 block of skeletal muscle excitation-contraction coupling

G proteins interacting with dihydropyridine receptors (DHPR) in transverse tubules (TT) of skeletal muscle may have a role in skeletal excitation-contraction (EC) coupling. The aim of this study was to determine the effects of G protein-specific nucleotides [guanosine 5'-O-(3-thiotriphosphate) (GTP gammaS) and guanosine 5'-O-(2-thiodiphosphate) (GDP betaS)] on the EC coupling mechanism in the presence of D-600, an agent that blocks EC coupling by immobilizing the voltage-sensing subunit of the DHPR in its inactivated state. By use of the mechanically peeled single-fiber preparation from rabbit adductor magnus skeletal muscle, 50 microM GTP gammaS and 500 microM GDP betaS were applied with the fiber in a D-600-induced state of blocked EC coupling. Neither nucleotide served as an independent stimulus for sarcoplasmic reticulum (SR) Ca2+ release when added to the TT polarizing bath under conditions of D-600 block. The presence of GTP gammaS or GDP betaS during a complete EC coupling cycle removed the D-600 block of EC coupling, despite continuous bath D-600. After the nucleotides were washed out, in the continued presence of D-600, the D-600 block of EC coupling was reestablished. In contrast, GTP gammaS added only during the period of TT depolarization under D-600 block did not remove the D-600 block of EC coupling, even though GTP gammaS did stimulate SR Ca2+ release. GTP gammaS had no effect on submaximum (0.5-1.0 mM) caffeine contractures and thus is unlikely to be acting through the Ca2+-induced Ca2+ release mechanism of the SR. These data suggest that the molecular binding site for GTP gammaS and GDP betaS is likely to be in the TT near the DHPR, perhaps on a G protein.

Steady-state twitch Ca2+ fluxes and cytosolic Ca2+ buffering in rabbit ventricular myocytes

Intracellular Ca2+ ([Ca2+]i) transients and transsarcolemmal Ca2+ currents were measured in indo 1-loaded isolated rabbit ventricular myocytes during whole cell voltage clamp to quantitate the components of cytosolic Ca2+ influx and to describe the dynamic aspects of cytosolic Ca2+ buffering during steady-state contraction (0.5 Hz, 22 degrees C). Sarcolemmal Ca2+ influx was directly measured from the integrated Ca2+ current (Ica) recorded during the clamp (158 +/- 10 attomoles; amol). Sarcoplasmic reticulum (SR) Ca2+ content was determined from the integrated electrogenic Na+/Ca2+ exchange current (Ix) induced during rapid application and sustained exposure of cells to caffeine to elicit the release of the SR Ca2+ load (1,208 +/- 170 amol). The mean steady-state SR Ca2+ load was calculated to be 87 +/- 13 microM (mumol/l nonmitochondrial cytosolic volume). Ca2+ influx via Ica represented approximately 14% of the stored SR Ca2+ and 23% of the total cytosolic Ca2+ flux during a twitch (47 +/- 6 microM). Comparison of electrophysiologically measured Ca2+ fluxes with Ca2+ transients yields apparent buffering values of 60 for caffeine contractures and 110 for twitches (delta Ca2+ total/delta Ca2+ free). This is consistent with the occurrence of "active" buffering of cytosolic Ca2+ by SR Ca2+ uptake during the twitch.

Two sites of action for LCB29 (idrocilamide) in depressing mechanical tension of rat soleus muscle fibers?

The effects of 50 μM LCB29 (idrocilamide) were tested on depolarization-induced and caffeine contractures of rat soleus muscle fibers. When applied intracellularly by free diffusion in cut-end voltage-clamped fibers, LCB29 decreased tension amplitude by about 25%. The same amount of inhibition by LCB29 was observed on contractures induced by 6 mM caffeine. The drug did not affect the repriming of caffeine contractures, indicating that internal recycling of calcium was not affected. The voltage-dependent inactivation of tension was facilitated by external application of LCB29. This effect was calcium dependent, so that the greater the external calcium concentration, the greater the drug effectiveness. The spontaneous relaxation of K+ contractures was also accelerated by LCB29. It is concluded that LCB29 acts intracellularly by decreasing sarcoplasmic reticulum calcium release and externally by facilitating the voltage-dependent inactivation of the voltage sensor for excitation–contraction coupling.Key words: myorelaxants, skeletal muscle, sarcoplasmic reticulum calcium release, voltage sensor.