Expression of mRNA for plasminogen activators and protease nexin‐1 in innervated and denervated mouse skeletal muscle

Abstract

Plasminogen activators (urokinase-type, u-PA and tissue-type, t-PA) are serine proteases that have been suggested to play important roles in synaptic remodeling. The enzymatic activity of u-PA in particular has previously been shown to increase dramatically after denervation of skeletal muscle. Using (32)P-labeled riboprobes and Northern blots the expression of mRNA for u-PA, t-PA and the inhibitor protease nexin-1 (PN-1) has been studied in innervated and 1-10-days denervated hind-limb muscle from mouse. Using RNA extracted from innervated and 6-days-denervated mouse hemidiaphragm muscles the expression of these mRNAs has also been investigated in synaptic and extrasynaptic muscle regions. For both u-PA and t-PA the observed autoradiographic signals were similar for RNA extracted from innervated and denervated leg muscles. The signals were also similar for RNA extracted from perisynaptic and extrasynaptic regions of hemidiaphragm muscle but u-PA signals were lower in denervated than in innervated hemidiaphragm. No such difference was observed for t-PA. PN-1 mRNA levels were also found to decrease after denervation in the hemidiaphragm but no substantial decrease was observed in denervated hind-limb muscles. No difference was observed between PN-1 expression in perisynaptic and extrasynaptic regions. The effect of denervation on PA enzymatic activity in skeletal muscle is therefore likely to be mediated at some post-transcriptional level.

Effects of skeletal muscle denervation on potency of rocuronium

“…Developing mouse denervation model according to previous reports [8,9], we successfully acutely isolated FDB muscle cells of mouse. We took an ACh concentration of 30 M as the agonist concentration.…”

Galectin-1 expression in innervated and denervated skeletal muscle

“…Denervation of the left hind-limb or the left hemidiaphragm was performed by sectioning and removing a few mm of the sciatic nerve or phrenic nerve. Six days (RNA extraction) or 6, 13 or 21 days (protein extraction) after denervation, muscles were harvested as described previously [13]. The experimental procedures were approved by the Ethical Committee for Animal Experiments at Linköping University, Sweden.…”

“…RNA extraction from the left hemidiaphragms of innervated or denervated mice was performed as described previously [13]. Muscle regions from 7 denervated (hypertrophic) or 16-20 innervated left hemidiaphragms were pooled.…”

“…Muscle regions from 7 denervated (hypertrophic) or 16-20 innervated left hemidiaphragms were pooled. Gastrocnemius, soleus, anterior tibial and extensor digitorum longus muscles from innervated or denervated hind-limbs were pooled and processed for RNA extraction as described previously [13].…”

“…Cloning of a galectin-1 complementary DNA fragment A complementary DNA (cDNA) fragment to be used for the synthesis of a labelled complementary RNA probe was amplified and cloned as described previously [13]. Gal1 cDNA was obtained with primers (forward: 5'-TGT GTA ACA CCA AGG AAG ATG G and reverse: 5'-CTG CCT TTA TTG AGG GCT ACA G) designed to amplify a 263-bp cDNA fragment corresponding to nucleotides 222-484 of the mouse Gal1 mRNA sequence (GenBank Accession number X15986, [14]).…”