Aplicación

Because of the massive induction of amphiregulin in PCLS after the slicing process, different inhibitors were used to investigate the mechanism of amphiregulin induction.

The highest upregulation could be observed 4h after the slicing process. Therefore inhibitors were added during the slicing process and for the next 4h of incubation.

The aim was to reduce the maximum induction 4h after the slicing process to possibly diminish the overall induction of amphiregulin. Without inhibition the increase in amphiregulin after 24h is still too high to re-induce it by stretch or to differentiate stretch-induction from the basal level.

4.3.3.1. Influence of tyrosine kinases

The specific tyrosine kinase inhibitor genistein and the selective Src-tyrosine-kinase inhibitor PP2 were used to examine the influence of tyrosine kinases on amphiregulin induction (Fig. 4.16). Genistein inhibited amphiregulin induction at 4h after the slicing process. After 24h there was no difference between solvent and genistein-treated PCLS. PP2 significantly reduced amphiregulin induction after 4h compared to solvent-treated PCLS. After 24h there was no more difference between solvent-solvent-treated and PP2-treated PCLS.

Figure 4.16: Amphiregulin expression in PCLS 4h and 24h after the slicing process treated with 100 µM of the tyrosine-kinase-inhibitor Genistein (A) and 10 µM of the Src-tyrosine-kinase inhibitor PP2 (B). Solvent-treated PCLS are illustrated as black columns and inhibitor-treated PCLS as grey columns.

Data are given as mean ± SD, n = 6 – 7 (Genistein) and 3 (PP2). Data was Box-Cox transformed and analysed by the Tukey Hsd test, p-values < 0.05 were considered significantly different (*); n.s. = non significant.

A B

These data indicate a role of tyrosine kinases in amphiregulin induction, possibly by Src-tyrosine kinases. After 4h a reduction of amphiregulin was observed, which was not seen 24h after the slicing process.

4.3.3.2. Influence of Sphingolipids

Sphingolipids, derived from the plasma membrane, are known to play an important role in the lungs (269). Acid sphingomyelinase generates ceramide, a constituent of ceramide-rich microdomains and a second messenger. Stress is well known to activate the acid sphingomyelinase, and therefore PCLS were treated with imipramine, an inhibitor of the acid sphingomyelinase pathway. Furthermore, PCLS were treated with sphingosine-1-phosphate that exerts protective effects in several insults (Fig 4.17). Again treatment was maintained during and for 4h after the slicing process.

No influence of the inhibition of the acid shingomyelinase was observed, neither 4h nor 24h after the slicing process. Treatment with sphingosine-1-phosphate increased the amphiregulin induction significantly 4h after the slicing process. There may be an activating effect on amphiregulin induction by sphingosine-1 phophate, which seems to be independent from acid sphingomyelinase.

4.3.3.3. Influence of calcium and ion-channels

Lanthanum chloride is used to block divalent cation channels, mainly calcium channels, and has been used as an unspecific inhibitor of stretch-activated ion channels (114; 227). Another selective calcium channel inhibitor is ruthenium red,

Figure 4.17: Amphiregulin RNA expression in PCLS treated with either 10 µM imipramine (A) or 1.32 µM sphingosine-1-phosphate (B). RNA expression was measured 4h and 24h after the slicing process. Solvent-treated PCLS are illustrated as black columns and inhibitor-treated PCLS as grey columns. Data are given as mean ± SD, n = 3. Data was Box-Cox transformed and analysed by the Tukey Hsd test, p-values < 0.05 were considered significantly different (*); n.s. = non significant.

A B

especially blocking TRPV channels and the ryanodine receptor (96; 306). As another approach, calcium in the slicing and incubation medium was reduced to 10 % of the normal amount (4.18). The treatments were applied during the slicing process and 4h thereafter. Reduced extracellular calcium slightly reduced amphiregulin induction after the slicing process, but this was not significant. 24h after the slicing process there was no difference in amphiregulin induction compared to control PCLS. No effect on amphiregulin induction was observed by treatment with lanthanum chloride (Fig. 4.19 B). Ruthenium red increased amphiregulin induction significantly 24h after the slicing process (Fig. 4.19 A).

Figure 4.18: A m p h i r e g u l i n induction in PCLS sliced and incubated in low calcium medium (grey columns, calcium reduced to 10 % of the normal amount) 4 and 24h after the slicing process. Control PCLS incubated in normal medium are illustrated as black columns.

Data are given as mean ± SD, n = 3. Data was Box-Cox transformed and analysed by the Tukey Hsd test, n.s. = non significant.

Figure 4.19: Amphiregulin induction in PCLS sliced and incubated for 4h with either 20 µM ruthenium red (A) or 100 µM lanthanum chloride (B), shown as grey columns. Control PCLS incubated in normal medium are illustrated as black columns. Data are given as mean ± SD, n = 3. Data was Box-Cox transformed and analysed by the Tukey Hsd test, p-values < 0.05 were considered significantly different (*); n.s. = non significant.

A B

In general reduced calcium does not appear to play a major role in amphiregulin induction, which does not seem to be mediated by store operated calcium channels, TRPV, or the ryanodine receptor.

4.3.3.4. Influence of the cytoskeleton

To investigate, whether the cytoskeleton plays a role in mediating the induction of amphiregulin, different inhibitors were used during the slicing process and 4h after it.

Y27632 is an inhibitor of the rho-kinase, which is involved in the organisation of the actin cytoskeleton. Besides inhibiting the Rho-kinases, cytochalasin D, a fungal toxin, which inhibits actin polymerisation, was employed. In addition, myosin light chain phosphorylation was blocked by ML-7. No decrease in amphiregulin induction was found after treatment with Y27632 (Fig. 4.20).

ML-7 and cytochalasin D both inhibited the maximum induction 4h after the slicing process (Fig. 4.21). In case of ML-7 this reduction was significant. Therefore an involvement of the myosin light chain kinase and the actin filaments is probable.

Figure 4.20: PCLS were treated with 10 µM Y27632 (grey columns) and amphiregulin RNA expression was compared to control (black columns) PCLS after 4 and 24h. Data are given as mean ± SD, n = 3. Data was Box-Cox transformed and analysed by the Tukey Hsd test, n.s. = non significant.