Inositol 1,4,5-trisphosphate: a possible chemical link in excitation-contraction coupling in muscle.

Abstract

The role of inositol 1,4,5-trisphosphate (InsP3) in excitation-contraction coupling in skeletal muscle was investigated by several methods. The following results were obtained. InsP3 is released by electrical stimulation of muscles. Exogenous InsP3 releases calcium from skinned muscle fibers at relatively high doses under normal conditions but does so at very low concentrations when blockers of the InsP3 5-phosphatase are present. Blockers of InsP3 release are effective blockers of calcium transients elicited by electrical stimulation of muscle fibers. It is proposed that InsP3 acts as a chemical second messenger between transverse (T)-tubular membrane depolarization and calcium release from the sarcoplasmic reticulum in skeletal muscle.One of the outstanding questions in muscle physiology is the mechanism of excitation-contraction coupling in vertebrate skeletal muscle. Electrical depolarization of the transverse (T) tubular system membranes somehow triggers release of Ca2+ ions from the sarcoplasmic reticulum (SR), which in turn activates the generation of tension (1-3). Though the T-tubule and SR are only some 100-200 A apart at the T/SR junctions, their membranes and luminal spaces do not seem to be physically continuous (4). How is the excitatory signal communicated from one structure to the other? The major present hypothesis assumes that intramembrane charge movement in the T-tubule membrane is mechanically linked to the SR membrane, causing the opening of Ca2' channels (5, 6). No direct evidence for such remote-control valves has been obtained. We now wish to present evidence for a biochemical coupling mechanism, in which inositol 1,4,5-trisphosphate (InsP3) acts as a soluble internal transmitter. This hypothesis was first suggested by two analogies. The first is the homology between SR and endoplasmic reticulum, from which InsP3 is known to release Ca2+ in other tissues (7,8 formation and activity should block excitation-contraction coupling; i.e., prevent T-system depolarization from releasing Ca2+.We report here the results of experiments that suggest that InsP3 may meet all four criteria. MATERIALS AND METHODSThe experiments on tension development were performed on segments of mechanically or chemically skinned skeletal muscle fibers isolated from the semitendinosus muscle of the frog Rana catesbeiana. Chemical skinning was performed by exposing muscle fibers for 10-20 min to saponin (20-50 ,ug/ml) (14) added to the relaxing solution described below. Mechanical skinning consisted of carefully splitting a segment of muscle fiber immersed in relaxing solution. Short segments (5-8 mm long) of skinned fibers were attached to a fixed hook and to a high sensitivity capacitive tension transducer. Tension was recorded on both chart paper and FM tape. The relaxing solution contained 110 mM potassium aspartate, 20 mM potassium Mops, 5 mM phosphocreatine, 100 ,uM EGTA, 3 mM ATP, and 3 mM MgSO4. The ATP and Mg2+ concentrations were adjusted in order to set the concentration offree Mg2+ at chosen levels. ...

Anatomical distribution of voltage-dependent membrane capacitance in frog skeletal muscle fibers.

“…This is in good agreement with our calculated density of q~ charge movements. Alternatively, the voltage-sensor in the transverse tubule membrane could be a molecule that produces or releases a transmitter, perhaps inositol 1,4,5-triphosphate (Vergara et al, 1985), which would then act to open sarcoplasmic reticulum calcium channels. The observation that 0.5 mM tetracaine apparently inhibits phosphatidylinositol phosphorylation in transverse tubule membranes isolated from frog skeletal muscles is in agreement with this idea (Hidalgo et al, 1986).…”

Influence of polyamines on membrane functions

“…This was indeed found to be the case, at least in vitro. It was shown that physiological concentrations of spermine and spermidine inhibited a phospholipase C-dependent hydrolysis of phosphoinositides in rat brain (Eichberg et al, 1981) and in platelets (Nahas & Graff, 1982) (Israels et al, 1986 Vergara et al (1985) concluded that spermine was able to induce a blockage of the Ca2" signals in electrically stimulated muscle fibres by interfering with the formation of Ins(1,4,5)P. Ins( 1 ,4,5)P3 5-phosphatase is a key enzyme which metabolizes the second messenger into the inactive Ins(1,4)P2 and thus terminates its action on Ca2" mobilization. This enzyme, which is predominantly particulate and associated with the plasma membrane, was found blocked by spermine at near physiological concentrations (Seyfred et al, 1984).…”

Structural and functional aspects of calcium homeostasis in eukaryotic cells

“…The evidence in favour of Ins(1,4,5)P3 may be summarized as follows: (a) Ins(1,4,5)P3 releases Ca2 + both from isolated SR terminal cisternae [234] and from skinned fibre preparations [234, 235, 241 -2441; (b) the entire machinery for the synthesis of the Ins(1,4,5)P3 precursor PtdIns(4,5)P2, Ins( 1 ,4,5)P3 formation (G-proteindependent phospholipase C) and Ins(1 ,4,5)P3 degradation [Ins(l ,4,5)P3 5-phosphatase] is present in the plasma membrane of striated muscles [245][246][247][248] and in a few cases, a selective enrichment of these synthetic and metabolic pathways has been found in T-tubules [245, 2481; (c) Ins(1,4,5)P3 levels increase dramatically upon electrical stimulation of skeletal muscles [235]; (d) Ins(1 ,4,5)P3, at micromolar concentrations, increases the opening probability of the Ca2 +-release channel of the SR after vesicle fusion with lipid bilayers [249]. A number of objections have been raised against the physiological role of Ins(1,4,5)P3 as a mediator in EC coupling in striated muscle, the most important being: (a) the release of Ca2 + from skinned skeletal muscle fibers activated by photohydrolyzed Ins(1 ,4,5)P3 is orders of magnitude slower than that observed under physiological conditions [250]; (b) the formation of Ins(1,4,5)P3 in electrically stimulated cells is observed only after tetanus [235]; (c) fiber contraction is not blocked by heparin, a known inhibitor preventing Ins(1 ,4,5)P3 binding to its receptor in smooth muscle and in non-muscle cells [251]. However, a high rate of contractions can be obtained by pressure injection of Ins(1,4,5)P3 [252] and heparin has no effect on the Ins(1 ,4,5)P3-induced contraction and Ca2+ release in skinned skeletal muscle fibers [253].…”