Volume 124, Issue 5, Pages 1476-1487 (May 2003) Involvement of integrins and Src in tauroursodeoxycholate-induced and swelling- induced choleresis  Dieter Häussinger, Anna Kordelia Kurz, Matthias Wettstein, Dirk Graf, Stephan Vom Dahl, Freimut Schliess  Gastroenterology  Volume 124, Issue 5, Pages 1476-1487 (May 2003) DOI: 10.1016/S0016-5085(03)00274-9

Figure 1 Effects of TUDC on the activities of p38MAPK, ERK-1, FAK, Src, and Lck in perfused rat liver. Livers were perfused in the presence of 100 μmol/L [3H]TC. After a preperfusion period of 20 minutes (t = 0 minute), TUDC (20 μmol/L) was added for another 30 minutes to the perfusion medium. Liver samples were taken after the time periods indicated. Protein kinase activities were determined as described in Materials and Methods and analyzed by densitometry. Protein kinase phosphorylations are given relative to those found at t = 0 minute. The protein levels of total p38MAPK, total ERK1/2, total FAK, or total Src were unchanged. (A) Representative immunoblots from a series of 3–5 independent perfusion experiments. (B) Statistical analysis. Statistically significant differences compared with t = 0 minute are indicated (∗P < 0.05). Gastroenterology 2003 124, 1476-1487DOI: (10.1016/S0016-5085(03)00274-9)

Figure 2 Effect of PP-2 and the integrin-inhibitory peptide GRGDSP on TUDC-induced activation of p38MAPK, ERK-1, FAK, and Src. Livers were perfused in the presence of 100 μmol/L [3H]TC. After a preperfusion period of 20 minutes (t = 0 minute), TUDC (20 μmol/L) was added for another 30 minutes to the perfusion medium. Liver samples were taken at the time points indicated. PP-2 (250 nmol/L) or GRGDSP (10 μmol/L) were added to the perfusion medium 30 minutes before the addition of TUDC. Protein kinase phosphorylations were determined as described in Materials and Methods and analyzed by densitometry. Protein kinase phosphorylations are given relative to the densities found at t = 0 minute. The protein levels of total p38MAPK, total ERK1/2, total FAK, or total Src were unchanged. Data are from 3–5 independent perfusion experiments and are given as means ± SEM. (A) Representative immunoblot for the effect of TUDC in the presence of PP-2 (250 nmol/L). (B) Representative immunoblot for the effect of TUDC in the presence of GRGDSP (10 μmol/L). (C) Time courses of TUDC effects: closed circles, no inhibitor; open squares, PP-2 present; open triangles, GRGDSP present. Statistical significant differences compared with t = 0 minute are indicated (∗P < 0.05). Gastroenterology 2003 124, 1476-1487DOI: (10.1016/S0016-5085(03)00274-9)

Figure 3 Effect of Src inhibition by (A) PP-2 and (B) the integrin-inhibitory peptide GRGDSP on the TUDC-induced stimulation of TC excretion in perfused rat liver. Livers were perfused with a medium containing 100 μmol/L [3H]TC. TUDC (20 μmol/L) and inhibitors were added at the time points indicated. Data are given as means ± SEM from 3–5 different experiments. (A) The Src inhibitor PP-2 (250 nmol/L; open circles) completely abolishes the choleretic effect of TUDC on TC excretion, which is found in the absence of the Src inhibitor (closed circles). PP-3 (250 nmol/L), an analogue without inhibitory effect on Src, had no significant effect on TUDC-induced choleresis (not shown). (B) The integrin-inhibitory peptide GRGDSP (10 μmol/L; open squares), but not its inactive analogue GRGESP (10 μmol/L; closed squares), abolishes the choleretic response to TUDC. GRGESP by itself had no effect on TUDC-induced stimulation of TC excretion (compare with A). Gastroenterology 2003 124, 1476-1487DOI: (10.1016/S0016-5085(03)00274-9)

Figure 4 Effects of TUDC on the activities of p38MAPK, ERK-1, FAK, PKB, and Src in perfused rat liver. Livers were perfused in the absence of TC. After a preperfusion period of 20 minutes (t = 0 minute), TUDC (20 μmol/L) was added for another 30 minutes to the perfusion medium. Liver samples were taken after the time points indicated. Protein kinase phosphorylations were determined as described in Materials and Methods and analyzed by densitometry. Protein kinase phosphorylations are given relative to the activities found at t = 0 minute. The protein levels of total p38MAPK, total ERK1/2, total FAK, or total Src were unchanged. (A) Representative immunoblots from a series of 3–5 independent perfusion experiments. (B) Statistical analysis. Data are given as means ± SEM. Statistically significant differences compared with t = 0 minute are indicated (∗P < 0.05). Gastroenterology 2003 124, 1476-1487DOI: (10.1016/S0016-5085(03)00274-9)

Figure 5 Effect of (A) the integrin-inhibitory peptide GRGDSP and (B) Src inhibition on the TUDC-induced inhibition of proteolysis in perfused rat liver. Livers were prelabeled in vivo by intraperitoneal injection of 150 μCi of [3H]leucine, and [3H]label release in the effluent was monitored as a measure of hepatic proteolysis in liver perfusion experiments (for further details, see Materials and Methods). Under control conditions in the absence of effectors, the release of [3H] label from the liver was set to 100%. TUDC (20 μmol/L) and inhibitors were added at the time points indicated. Data are given as means ± SEM from 3–5 different experiments. (A) The Src inhibitor PP-2 (250 nmol/L; open circles) abolishes the antiproteolytic effect of TUDC, which is observed in the absence of PP-2 (closed circles). (B) TUDC induces an inhibition of proteolysis in control experiments (closed squares). In the presence of the integrin-inhibitory peptide GRGDSP (10 μmol/L; open squares), the antiproteolytic effect of TUDC is strongly blunted. Gastroenterology 2003 124, 1476-1487DOI: (10.1016/S0016-5085(03)00274-9)

Figure 6 Effect of hypo-osmotic exposure on the activities of p38MAPK, ERK-1, FAK, PKB, and Src in perfused rat liver. Livers were perfused with a medium containing 100 μmol/L [3H]TC. After a preperfusion with normo-osmotic medium (305 mOsmol/L), hypo-osmotic conditions (225 mOsmol/L) were instituted (t = 0 minute). Liver samples were taken after the time points indicated. Protein kinase phosphorylations were determined as described in Materials and Methods and analyzed by densitometry. Protein kinase phosphorylations are given relative to the activities found at t = 0 minute. Protein levels of total p38MAPK, total ERK1/2, total FAK, or total Src were unchanged. (A) Representative immunoblots from a series of 3–5 independent perfusion experiments. (B) Statistical analysis. Data are given as means ± SEM. Statistical significance is indicated (∗P < 0.05). Gastroenterology 2003 124, 1476-1487DOI: (10.1016/S0016-5085(03)00274-9)

Figure 7 Effect of Src inhibition by (A) PP-2 and (B) the integrin-inhibitory peptide GRGDSP on the hypo-osmotic stimulation of TC excretion in perfused rat liver. Livers were perfused with a medium containing 100 μmol/L [3H]TC. Hypo-osmolarity was instituted and inhibitors were added at the time points indicated. Data are given as means ± SEM from 4–6 different experiments. (A) Hypo-osmolarity stimulates TC excretion into bile (closed squares). This stimulation is largely abolished in the presence of the Src inhibitor PP-2 (250 nmol/L; open squares). Note that PP-2 has no effect on TC excretion per se. (B) The integrin-inhibitory peptide GRGDSP (10 μmol/L; open circles), but not its inactive analogue GRGESP (10 μmol/L; closed circles), abolishes the choleretic response to TUDC. Gastroenterology 2003 124, 1476-1487DOI: (10.1016/S0016-5085(03)00274-9)

Figure 8 Effect of Src inhibition by PP-2 and the integrin-inhibitory peptide GRGDSP on the hypo-osmotic p38MAPK activation in 48-hour cultured rat hepatocytes. Forty-eight-hour cultured rat hepatocytes were preincubated with PP-2 (10 μmol/L) or GRGDSP (20 μmol/L). Thereafter, hepatocytes were exposed for another 5 minutes to 205 mOsmol/L or 305 mOsmol/L (control). Representative results from a series of 3–5 independent perfusion experiments are shown. Both PP-2 and GRGDSP prevent the hypo-osmotic p38MAPK activation. Gastroenterology 2003 124, 1476-1487DOI: (10.1016/S0016-5085(03)00274-9)

Figure 9 Hypothetical scheme on TUDC-induced signaling toward bile acid excretion in rat liver. The scheme is based on signal-transduction and inhibitor studies with respect to the TUDC-induced stimulation of bile acid excretion in primary rat hepatocytes and perfused rat liver.6,10,11,19 TUDC activates via integrins FAK and Src, which mediate downstream signaling toward MAPK. Gastroenterology 2003 124, 1476-1487DOI: (10.1016/S0016-5085(03)00274-9)