ABSTRACT
Duchenne muscular dystrophy is caused by deficiency of dystrophin and leads to progressive weakness. It has been proposed that the muscle degeneration occurring in this disease is caused by increased Ca2+ influx due to enhanced activity of cationic channels that are activated either by stretch of the plasma membrane (stretch-activated channels) or by Ca2+-store depletion (store-operated channels). Using both cytosolic Ca2+ measurements with Fura-2 and the manganese quench method, we show here that store-operated Ca2+ entry is greatly enhanced in dystrophic skeletal flexor digitorum brevis fibers isolated from mdx(5cv) mice, a mouse model of Duchenne muscular dystrophy. Moreover, we show for the first time that store-operated Ca2+ entry in these fibers is under the control of the Ca2+-independent phospholipase A2 and that the exaggerated Ca2+ influx can be completely attenuated by inhibitors of this enzyme. Enhanced store-operated Ca2+ entry in dystrophic fibers is likely to be due to a near twofold overexpression of Ca2+-independent phospholipase A2. The Ca2+-independent phospholipase A2 pathway therefore appears as an attractive target to reduce excessive Ca2+ influx and subsequent degeneration occurring in dystrophic fibers.
Subject(s)
Calcium Signaling/physiology , Muscle Fibers, Skeletal/enzymology , Muscle, Skeletal/enzymology , Muscular Dystrophy, Animal/chemically induced , Phospholipases A/metabolism , Anilides/pharmacology , Animals , Caffeine/pharmacology , Calcium Channels/metabolism , Calcium Signaling/drug effects , Group VI Phospholipases A2 , Ion Transport/drug effects , Manganese/metabolism , Melitten/pharmacology , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Models, Biological , Muscle Fibers, Skeletal/drug effects , Muscle, Skeletal/drug effects , Naphthalenes/pharmacology , Phospholipases A/antagonists & inhibitors , Phospholipases A2 , Potassium Chloride/metabolism , Pyrones/pharmacology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Thiadiazoles/pharmacologyABSTRACT
Agrin triggers signaling mechanisms of high temporal and spatial specificity to achieve phosphorylation, clustering, and stabilization of postsynaptic acetylcholine receptors (AChRs). Agrin transiently activates the kinase MuSK; MuSK activation has largely vanished when AChR clusters appear. Thus, a tyrosine kinase cascade acts downstream from MuSK, as illustrated by the agrin-evoked long-lasting activation of Src family kinases (SFKs) and their requirement for AChR cluster stabilization. We have investigated this cascade and report that pharmacological inhibition of SFKs reduces early but not later agrin-induced phosphorylation of MuSK and AChRs, while inhibition of Abl kinases reduces late phosphorylation. Interestingly, SFK inhibition applied selectively during agrin-induced AChR cluster formation caused rapid cluster dispersal later upon agrin withdrawal. We also report that a single 5-min agrin pulse, followed by extensive washing, triggered long-lasting MuSK and AChR phosphorylation and efficient AChR clustering. Following the pulse, MuSK phosphorylation increased and, beyond a certain level, caused maximal clustering. These data reveal novel temporal aspects of tyrosine kinase action in agrin signaling. First, during AChR cluster formation, SFKs initiate early phosphorylation and an AChR stabilization program that acts much later. Second, a kinase mechanism rapidly activated by agrin acts thereafter autonomously in agrin's absence to further increase MuSK phosphorylation and cluster AChRs.
Subject(s)
Agrin/pharmacology , Receptor Aggregation/drug effects , Receptors, Cholinergic/drug effects , Receptors, Cholinergic/metabolism , Agrin/administration & dosage , Agrin/metabolism , Animals , Binding Sites , COS Cells , Clone Cells , Enzyme Activation/drug effects , Mice , Myoblasts/drug effects , Myoblasts/metabolism , Phosphorylation , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-abl/metabolism , Proto-Oncogene Proteins c-fyn , Receptor Protein-Tyrosine Kinases/metabolism , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacology , Signal Transduction/drug effects , Synapses/metabolism , src-Family Kinases/chemistry , src-Family Kinases/metabolismABSTRACT
We examined the effect of neurons on oxytocin (OT) receptors (OTR) and OTR gene expression in cultured astrocytes. The addition of neuron-conditioned medium induced an increase of both OTR binding and OTR mRNA level. This effect was enhanced after the medium was boiled or acidified. As it is known that transforming growth factor-beta (TGF-beta) can be released from carrier proteins by acid or heat, TGF-beta1 and 2 were tested and found to induce an increase of OTR binding. Furthermore, TGF-beta antibody abolished the stimulatory effect of normal or acidified neuron-conditioned medium. Neurons added to cultured astrocytes without contact mimicked the stimulatory effect of the conditioned medium. In contrast, neurons added with contact, induced a decrease in OTR binding and an increase of mRNA level, whereas neuronal membranes induced a decrease of both OTR binding and mRNA levels. In conclusion, the present data demonstrate that in vitro, neurons are able to modulate astrocytic OTR expression at the level of both protein and mRNA. They stimulate this expression through their release of TGF-beta and inhibit it by the action of unknown membrane components.
