ryanodine and 1-2-dioctanoylglycerol

The contraction and relaxation of skeletal muscle is driven by release of Ca2+ from sarcoplasmic reticulum through the ryanodine receptor type 1 and extruding the ion from the cytosol by Ca2+ ATPases. Efficient refilling of the empty Ca2+ stores is essential for repetitive cycles of muscle contraction and relaxation, but not investigated in human skeletal muscle cells. Here we show that under conditions of selective depletion of the ryanodine-sensitive Ca2+ pool Ca2+ influx occurs in differentiated human skeletal muscle cells using the Ca2+ imaging technique. This Ca2+ influx is not due to permeation through the L-type Ca2+ channel and not observed under conditions of inhibited Ca2+ ATPase. The Ca2+ influx was visualised by quenching the intracellular fura2 signal with Mn2+ on single cell level and also using fluorescence photometry of cell suspensions. The Mn2+ influx was inhibited by the Ca2+ channel blockers La(3+) and SKF96356. The delineation of the signalling cascade leading to Ca2+ influx evoked by selective depletion of ryanodine sensitive Ca2+ stores showed that phospholipase C or protein kinase C were not involved. Interestingly, a Mn2+ influx was triggered by the cell-permeant analogue of diacylglycerol and further augmented by the application of RHC80267, a diacylglycerol lipase inhibitor. This signalling pathway could be attributed to the participation of a protein kinase C activity. However, Mn2+ influx evoked by selective depletion of ryanodine sensitive Ca2+ stores was not altered by RHC80267 or protein kinase C inhibitors. Using RT-PCR, correctly spliced mRNA fragments were detected corresponding to human transient receptor potential (TRPC) Ca2+ channels type 1, 3, 4 and 6. These data show that in skeletal muscle at least two independent mechanisms of Ca2+ influx exist. For Ca2+ influx triggered by the selective depletion of ryanodine sensitive Ca2+ stores we propose a phospholipase C independent coupling of ryanodine receptors to voltage insensitive Ca2+ channels.

Topics: Caffeine; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Cells, Cultured; Cyclohexanones; Diglycerides; Humans; Lipoprotein Lipase; Magnesium; Muscle Cells; Muscle, Skeletal; Nifedipine; Protein Kinase C; Reverse Transcriptase Polymerase Chain Reaction; Ryanodine; Ryanodine Receptor Calcium Release Channel; Time Factors; Type C Phospholipases

Local Ca2+ transients ("Ca2+ sparks") caused by the opening of one or the coordinated opening of a number of tightly clustered ryanodine-sensitive Ca(2+)-release (RyR) channels in the sarcoplasmic reticulum (SR) activate nearby Ca(2+)-dependent K+ (KCa) channels to cause an outward current [referred to as a "spontaneous transient outward current" (STOC)]. These KCa currents cause membrane potential hyperpolarization of arterial myocytes, which would lead to vasodilation through decreasing Ca2+ entry through voltage-dependent Ca2+ channels. Therefore, modulation of Ca2+ spark frequency should be a means to regulation of KCa channel currents and hence membrane potential. We examined the frequency modulation of Ca2+ sparks and STOCs by activation of protein kinase C (PKC). The PKC activators, phorbol 12-myristate 13-acetate (PMA; 10 nM) and 1,2-dioctanoyl-sn-glycerol (1 microM), decreased Ca2+ spark frequency by 72% and 60%, respectively, and PMA reduced STOC frequency by 83%. PMA also decreased STOC amplitude by 22%, which could be explained by an observed reduction (29%) in KCa channel open probability in the absence of Ca2+ sparks. The reduction in STOC frequency occurred in the presence of an inorganic blocker (Cd2+) of voltage-dependent Ca2+ channels. The reduction in Ca2+ spark frequency did not result from SR Ca2+ depletion, since caffeine-induced Ca2+ transients did not decrease in the presence of PMA. These results suggest that activators of PKC can modulate the frequency of Ca2+ sparks, through an effect on the RyR channel, which would decrease STOC frequency (i.e., KCa channel activity).

Topics: Animals; Basilar Artery; Calcium; Calcium Channels; Diglycerides; Enzyme Activation; In Vitro Techniques; Kinetics; Models, Biological; Muscle, Smooth, Vascular; Protein Kinases; Rats; Ryanodine; Tetradecanoylphorbol Acetate