ryanodine and 1-6-bis(cyclohexyloximinocarbonyl)hexane

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

In this study we have examined for the first time the signal transduction mechanisms involved in the generation of pressure-dependent myogenic tone in human small resistance arteries from the subcutaneous vascular bed.. Myogenic responses and the subcellular mechanisms involved in the generation of this response were studied on a pressure myograph.. Human subcutaneous resistance arteries constricted 14.1+/-1.1% in response to an increases in intraluminal pressure from 40 to 80 mmHg and a further 3.5+/-1.7% in response to the 80-120-mmHg pressure step. Ca(2+) depletion or nifedipine abolished this response, whereas BAY K 8644 increased this response to 20.6+/-2.1% (P<0.05, response vs. control). The phospholipase C inhibitor U-73122 reduced the myogenic response to 2.5+/-1.0% at 80 mmHg (P<0.01, response vs. control) and abolished it at 120 mmHg. Diacylglycerol lipase inhibition with RHC-80267 abolished all myogenic responses to pressure. The protein kinase C (PKC) activator phorbol 12,13-dibutyrate increased the maximal myogenic response to 20.9+/-1.8% (P<0.05, response vs. control), whereas the PKC inhibitor calphostin C abolished myogenic responses. These data show that the generation of pressure-dependent myogenic tone in human subcutaneous arteries is dependent on Ca(2+) influx via voltage operated Ca(2+) channels (VOCCs) and a concomitant requirement for the activation of phospholipase C (PLC), diacylglycerol, and PKC.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Aged; Analysis of Variance; Arteries; Caffeine; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium-Transporting ATPases; Cyclohexanones; Enzyme Inhibitors; Estrenes; Female; Humans; In Vitro Techniques; Indoles; Lipoprotein Lipase; Male; Middle Aged; Muscle, Smooth, Vascular; Naphthalenes; Nifedipine; Phorbol 12,13-Dibutyrate; Protein Kinase C; Pyrrolidinones; Ryanodine; Ryanodine Receptor Calcium Release Channel; Signal Transduction; Type C Phospholipases; Vascular Resistance