The somatic muscle of Ascaris suum is principally under the excitatory control of neuromuscular junction transmitter, acetylcholine (ACh). However, it has recently been shown that neuropeptides also play an important role in the motor-nervous system and one of these, AF3 (AVPGVLRFamide), also contracts muscle. The events which trigger contraction to ACh and AF3 would appear to be different, with ACh activating a nicotinic acetylcholine receptor whilst the response to AF3 is most likely to involve a G-protein coupled receptor negatively coupled to adenylate cyclase. In order to further elucidate differences in the cellular signalling pathways through which ACh and AF3 elicit muscle contraction, we investigated the actions of protein kinase C inhibitors, tamoxifen and chelerythrine, on the dorsal somatic muscle strip of A. suum. Contractions in response to 1 μ M AF3 were potentiated by 17% in the presence of 10 μ M tamoxifen ( P <0·05; n =8); however, contractions in response to 10 μ M ACh were markedly inhibited (tamoxifen IC 50 44±18 μ M ; n =6). Tamoxifen also blocked muscle cell depolarizations to 5 μ M ACh (IC 50 4±1 μ M ; n =6) and 1 μ M levamisole (IC 50 14±6 μ M ; n =4). This was unlikely to be a non-specific effect on the membrane as hyperpolarizations to 10 μ M GABA were unaffected (93% of control with 10 μ M tamoxifen; n =6; P >0·05). However, another inhibitor of mammalian protein kinase C, chelerythrine, did not affect the response either to ACh or AF3 ( n =6).
AF3 (AVPGVLRFamide) and AF4 (GDVPGVLRFamide) are endogenous RFamide-like peptides isolated from the parasitic nematode Ascaris suum . Here the actions of these peptides on the somatic musculature of Ascaris have been investigated and compared to the action of acetylcholine (ACh), the excitatory transmitter at the neuromuscular junction. ACh, AF3 and AF4 contracted muscle with EC 50 s of 13±1 μ M , 24±6 n M and 37±2 n M , respectively ( n =6). The muscle cells were depolarized by ACh (3 μ M ; 5·2±0·4 mV, n =42), AF3 (1 μ M ; 2·6±0·3 mV, n =19) and AF4 (1 μ M ; 3·3±0·4 mV, n =19). EC 50 s were 681±329 n M (AF3) and 901±229 n M (AF4), but an estimate could not be made for ACh due to muscle contraction at concentrations greater than 10 μ M . The depolarization to 3 μ M ACh was abolished by the nicotinic receptor antagonist mecamylamine (10 μ M ; n =5) but the responses to the peptides were not (111±7% and 108±17% with respect to control; n =5). The depolarization elicited by ACh was reduced to a greater extent by a 50% reduction in extracellular Na + concentration than the response to AF3 and AF4 ( P <0·02). Cobalt was more effective at blocking the AF3 and AF4 depolarizations than those to ACh. These observations suggest that AF3 and AF4 contract Ascaris muscle without an action at the Ascaris nicotinic receptor. Furthermore, the ionic mechanism through which AF3 and AF4 depolarize Ascaris muscle is different from that for ACh. ACh, AF3 and AF4 were also found to contract Ascaridia galli somatic muscle with EC 50 s of 13±3 μ M , 721±236 n M and 371±177 n M , respectively ( n =7). The muscle cells were depolarized by ACh (EC 50 =14±5 μ M , n =5), AF3 (EC 50 =5±3 μ M , n =4) and AF4 (EC 50 =10±5 μ M , n =4). Therefore the response to these peptides is not unique to Ascaris and they may subserve a functional role in the motor nervous system of parasitic nematodes.
Myofibroblastic-activated hepatic stellate cells are the major source of the collagen I-rich extracellular matrix in liver fibrosis but also produce matrix metalloproteinases, which remodel this protein. We have investigated the role of collagen I proteolysis in both regulating proliferation and maintaining the activated myofibroblastic phenotype of stellate cells in vitro. Compared with stellate cells plated on normal collagen I, those plated on a collagenase-resistant form of collagen I (r/r collagen) had reduced thymidine incorporation and proliferating cell nuclear antigen expression but increased p21 expression. Collagen I was shown to be rendered resistant to matrix metalloproteinases by artificial cross-linking in vitro using tissue transglutaminase exerted similar antiproliferative effects on stellate cells to r/r collagen. Of the stellate cell activation markers examined (tissue inhibitor of metalloproteinases-1, alpha-smooth muscle actin, matrix metalloproteinases-2 and -9, and procollagen I) only the last was decreased by culture on r/r collagen relative to normal collagen I. Antagonists of integrin alphavbeta3, an integrin reported to stimulate stellate cell proliferation, significantly inhibited adhesion, proliferation, and procollagen I synthesis of stellate cells plated on normal collagen I but had reduced effectiveness on these parameters in cells on r/r collagen. We conclude that proliferation of stellate cells is promoted by pericellular collagen I proteolysis acting via alphavbeta3 integrin. Cross-linking of collagen I by tissue transglutaminase, a process known to occur in chronic liver fibrosis, might not only increase its resistance to matrix metalloproteinases thereby inhibiting resolution of fibrosis but also functions to constrain the fibroproliferative process.
Collagen-I, which predominates in the neomatrix of fibrotic liver, regulates hepatocyte and hepatic stellate cell (HSC) phenotypes. Recovery from liver fibrosis is accompanied by hepatocyte regeneration, matrix degradation, and HSC apoptosis. Using mice bearing a mutated collagen-I gene (r/r mice), which confers resistance to collagenase degradation, we have investigated the hypothesis that collagen-I degradation is critical to HSC apoptosis and hepatocyte regeneration during recovery from liver fibrosis. During a 28-day recovery period after 8 wk of CCl4 treatment, wild-type (WT) livers had significantly (43%) decreased hydroxyproline (OHP) content. In r/r livers, however, OHP content remained elevated at peak fibrosis levels. Expressed markers of activated HSC (α-smooth muscle actin, collagen-I), elevated at peak fibrosis, dropped to control levels in WT livers after 28 days but remained raised in the r/r livers. Moreover, relative to WT livers, r/r livers had significantly reduced stellate cell apoptosis and hepatocyte regeneration during the recovery period. Using extracted collagen-I from each genotype as culture substrata, relative to r/r, we show that WT collagen-I promotes hepatocyte proliferation via stimulation of integrin αvβ3. Failure to degrade collagen-I critically impairs HSC apoptosis and may prevent the effective restoration of hepatocyte mass in liver fibrosis.
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