Epithelia from many tissues express protease-activated receptors (PARs) that play a major role in several different physiological processes. In this study, we examined their capacity to modulate IL-6, IL-8, and PGE2 production in both the A459 and BEAS-2B cell lines and primary human bronchial epithelial cells (HBECs). All three cell types expressed PAR-1, PAR-2, PAR-3, and PAR-4, as judged by RT-PCR and immunocytochemistry. Agonist peptides corresponding to the nascent N termini of PAR-1, PAR-2, and PAR-4 induced the release of cytokines from A549, BEAS-2B, and HBECs with a rank order of potency of PAR-2 > PAR-4 > PAR-1 at 400 μM. PAR-1, PAR-2, and PAR-4 also caused the release of PGE2 from A549 and HBECs. The PAR-3 agonist peptide was inactive in all systems tested. PAR-1, PAR-2, or PAR-4, in combination, caused additive IL-6 release, but only the PAR-1 and PAR-2 combination resulted in an additive IL-8 response. PAR peptide-induced responses were accompanied by changes in intracellular calcium ion concentrations. However, Ca2+ ion shutoff was ∼2-fold slower with PAR-4 than with PAR-1 or PAR-2, suggesting differential G protein coupling. Combined, these data suggest an important role for PAR in the modulation of inflammation in the lung.

In previous studies, we demonstrated that allergenic house dust mite proteases are potent inducers of proinflammatory cytokines from the respiratory epithelium, although the precise mechanisms involved were unclear. In this study, we investigated whether this was achieved through activation of protease-activated receptor (PAR)-1 or -2. Pretreatment of A549 respiratory epithelial cells with the clinically important cysteine protease allergen, Der p 1, ablated subsequent PAR-1, but not PAR-2 agonist peptide-induced IL-6 and IL-8 release. HeLa cells transfected with the plasmid coding for PAR-2, in contrast to PAR-1, released significant concentration of IL-6 after exposure to Der p 1. Exposure of HeLa cells transfected with either PAR-1/enhanced yellow fusion protein or PAR-2/enhanced yellow fusion protein to Der p 1 caused receptor internalization in the latter cells only, as judged by confocal microscopy with re-expression of the receptor within 120-min postenzyme exposure. Der p 1-induced cytokine release from both A549 and transfected HeLa cells was accompanied by changes in intracellular Ca2+ concentrations. Desensitization studies showed that Der p 1 pretreatment of the A549 cells resulted in the abolition of both trypsin- and PAR-2 agonist peptide-induced Ca2+ release, but not that induced by subsequent exposure to either thrombin or PAR-1 agonist peptide. These data indicate for the first time that the house dust mite allergen Der p 1-induced cytokine release from respiratory epithelial cells is, in part, mediated by activation of PAR-2, but not PAR-1.

Mutations in the skeletal muscle α-actin gene (ACTA1) cause a range of pathologically defined congenital myopathies. Most patients have dominant mutations and experience severe skeletal muscle weakness, dying within one year of birth. To determine mutant ACTA1 pathobiology, transgenic mice expressing ACTA1(D286G) were created. These Tg(ACTA1)(D286G) mice were less active than wild-type individuals. Their skeletal muscles were significantly weaker by in vitro analyses and showed various pathological lesions reminiscent of human patients, however they had a normal lifespan. Mass spectrometry revealed skeletal muscles from Tg(ACTA1)(D286G) mice contained ∼25% ACTA1(D286G) protein. Tg(ACTA1)(D286G) mice were crossed with hemizygous Acta1(+/-) knock-out mice to generate Tg(ACTA1)(D286G)(+/+).Acta1(+/-) offspring that were homozygous for the transgene and hemizygous for the endogenous skeletal muscle α-actin gene. Akin to most human patients, skeletal muscles from these offspring contained approximately equal proportions of ACTA1(D286G) and wild-type actin. Strikingly, the majority of these mice presented with severe immobility between postnatal Days 8 and 17, requiring euthanasia. Their skeletal muscles contained extensive structural abnormalities as identified in severely affected human patients, including nemaline bodies, actin accumulations and widespread sarcomeric disarray. Therefore we have created valuable mouse models, one of mild dominant ACTA1 disease [Tg(ACTA1)(D286G)], and the other of severe disease, with a dramatically shortened lifespan [Tg(ACTA1)(D286G)(+/+).Acta1(+/-)]. The correlation between mutant ACTA1 protein load and disease severity parallels effects in ACTA1 families and suggests altering this ratio in patient muscle may be a therapy for patients with dominant ACTA1 disease. Furthermore, ringbinden fibres were observed in these mouse models. The presence of such features suggests that perhaps patients with ringbinden of unknown genetic origin should be considered for ACTA1 mutation screening. This is the first experimental, as opposed to observational, evidence that mutant protein load determines the severity of ACTA1 disease.

The sulfonic amino acid taurine is found in high concentrations in many mammalian excitable cells and is reported to have a variety of functions including osmoregulation, modulation of neuronal excitability, antioxidation and control of Ca 2+ homeostasis (Huxtable, 1992).Taurine is also found in high levels in skeletal muscle (Chesney et al. 1986; Nieminen et al. 1988; Turner et al. 1994). The taurine content in skeletal muscle is reported to vary between muscle type and species. In the rat, the slowtwitch soleus muscle is reported to have twice the taurine content (33 µmol (g wet weight) _1 ) of the fast-twitch extensor digitorum longus (EDL) muscle (17 µmol (g wet weight)_1 ; Iwata et al. 1986). In the horse, slow-twitch type I fibres have been reported to have a high taurine content, while in fast-twitch type IIb fibres, taurine was reported to be undetectable (Dunnett et al. 1992). Recent studies suggest that the taurine content within skeletal muscle may also vary markedly between fibres in the same muscle. Wide differences in the taurine immunoreactivity of individual fibres within the same skeletal muscle have been reported for a number of species, including the rat and the cat (Quesada et al. 1993; Lobo et al. 2000). In the cat soleus muscle, the taurine immunoreactivity was reported to be high and relatively homogeneous (Quesada et al. 1993).The reason for the heterogeneity in the taurine content of mammalian skeletal muscle is unknown. Taurine is actively accumulated by most cells via a Na + -dependent high affinity taurine transporter, and this taurine transporter has been shown to be highly expressed in skeletal muscle (Ramamoorthy et al. 1994). Taurine release has also been demonstrated in many cell types, and one of the main pathways is volume activated (via hyposmotic conditions), and is thought to involve specific anion channels (Perlman & Goldstein, 1999). Hyposmotic conditions and pathological factors such as ischaemia have been shown to lead to taurine release in cardiac myocytes (Kramer et al. 1981) and neurones (Schouboe & Pasantes-Morales, 1992; Saransaari & Oja, 1998) by mechanisms that are poorly understood.In skeletal muscle, there is evidence that contractile activity may induce changes in myoplasmic taurine content. Chronic sciatic nerve stimulation (100 Hz) has been shown to increase the taurine content of rat EDL muscle, and decrease the taurine content in rat soleus muscle (Kim et al. 1986). After neural stimulation, the proportion of taurine-positive skeletal muscle fibres (determined by immunoreactivity measurements) has been reported to fall in the cat (Quesada et al. 1993). We examined the effect of taurine on depolarisation-induced force responses and sarcoplasmic reticulum (SR) function in mechanically skinned skeletal muscle fibres from the extensor digitorum longus (EDL) of the rat. Taurine (20 m) produced a small but significant (P < 0.01) decrease in the sensitivity of the contractile apparatus to Ca 2+ (increase in the [Ca 2+ ] corresponding to 50 % of maximum force...