Reduced nitric oxide production by endothelial cells in cirrhotic rat liver: Endothelial dysfunction in portal hypertension  Don C. Rockey, John J. Chung  Gastroenterology  Volume 114, Issue 2, Pages 344-351 (February 1998) DOI: 10.1016/S0016-5085(98)70487-1 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 ecNOS mRNA expression in hepatic cell types. Hepatic cell types were isolated as described in Materials and Methods. RNase protection assay was performed using RNA from freshly isolated cells with radiolabeled ecNOS cRNA (5 μg of total RNA). A representative autoradiogram is shown (n = 4). Positive and negative control samples were from whole rat aorta (AE) and transfer RNA (tRNA), respectively. H, hepatocyte; K, Kupffer cell; SC, stellate cell; SE, sinusoidal endothelial cell; AE, aortic endothelium. Gastroenterology 1998 114, 344-351DOI: (10.1016/S0016-5085(98)70487-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 ecNOS expression in SECs after obstructive liver injury. Bile duct ligation was performed as described in Materials and Methods. SECs were isolated at the number of days after ligation shown, and RNase protection assay using radiolabeled ecNOS cRNA was performed with total RNA (5 μg) from freshly isolated cells. An autoradiogram from a representative RNase protection assay is shown. S-14 is an internal standard RNA used to quantitate loaded RNA. Specific bands were scanned, quantitated, and normalized to the signal for S-14 mRNA. There were no statistical differences in the amounts of ecNOS mRNA at any time point after bile duct ligation vs. normal (n = 3). Gastroenterology 1998 114, 344-351DOI: (10.1016/S0016-5085(98)70487-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 ecNOS expression in SECs after toxin-induced liver injury. Hepatic injury and cirrhosis were induced with CCl4 administered by gavage as described in Materials and Methods. SECs were isolated 7 days after the number of weekly doses of CCl4 shown. RNase protection assay was performed using radiolabeled ecNOS cRNA with total RNA (5 μg) from freshly isolated cells. An autoradiogram from a representative RNase protection assay is shown. Specific bands were scanned, quantitated, and normalized to the signal for S-14 mRNA (S-14 is an internal standard RNA used to quantitate loaded RNA). There were no statistical differences in the amounts of ecNOS mRNA at any time point after CCl4 vs. normal (n = 3). Gastroenterology 1998 114, 344-351DOI: (10.1016/S0016-5085(98)70487-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 NOS activity is reduced in SECs from cirrhotic liver (▨) compared with normal liver (■). Liver injury, cirrhosis, and portal hypertension were induced with CCl4 administered by gavage (10 doses) as described in Materials and Methods. SECs were isolated and placed in culture (density, 2.0 × 106/cm2) for 18 hours to allow adherence. NOS activity ([3H]L-arginine conversion to [3H]L-citrulline) in the presence or absence of NMMA (100 μmol/L) was measured in SECs as described in Materials and Methods. *P < 0.05 vs. normal; ‡P < 0.05 vs. control (n = 4). Gastroenterology 1998 114, 344-351DOI: (10.1016/S0016-5085(98)70487-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Reduced accumulation of nitrite by SECs from cirrhotic liver (▨) compared with normal liver (■). Liver injury and cirrhosis were as in Figure 4. SECs were isolated and placed in culture for 24 hours to allow adherence and nitrite accumulation. Nitrite was quantitated as described in Materials and Methods and expressed as total nitrite (per 24 hours per total protein within the monolayer). *P < 0.05 vs. normal; ‡P < 0.05 vs. control (n = 4). Gastroenterology 1998 114, 344-351DOI: (10.1016/S0016-5085(98)70487-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Reduced release of NO by SECs from cirrhotic liver (▨) compared with normal liver (■). Liver injury and cirrhosis were as in Figure 4. SECs were isolated and placed in culture for 24 hours to allow adherence. cGMP was quantitated as described in Materials and Methods in either (A) SECs themselves or in (B) RFL-6 cells after exposure to SEC conditioned medium. NMMA (100 μmol/L) and ODQ (5 μmol/L) were added at the time of plating to certain cultures. *P < 0.05 vs. normal; ‡P < 0.05 vs. control (n = 6). Gastroenterology 1998 114, 344-351DOI: (10.1016/S0016-5085(98)70487-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Reduced release of NO by SECs from cirrhotic liver (▨) compared with normal liver (■). Liver injury and cirrhosis were as in Figure 4. SECs were isolated and placed in culture for 24 hours to allow adherence. cGMP was quantitated as described in Materials and Methods in either (A) SECs themselves or in (B) RFL-6 cells after exposure to SEC conditioned medium. NMMA (100 μmol/L) and ODQ (5 μmol/L) were added at the time of plating to certain cultures. *P < 0.05 vs. normal; ‡P < 0.05 vs. control (n = 6). Gastroenterology 1998 114, 344-351DOI: (10.1016/S0016-5085(98)70487-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions