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119 FREE AND CONJUGATED PLASMA CATECHOLAMINES IN NORMAL RATS AND IN RATS WITH PORTAL HYPERTENSION DUE TO PORTAL VEIN STENOSIS C. Gaudin, G. Ru~et, F. Selz, C. Girod, JL. Cuche, D. Lebrec INSERM U 27, NSpital Foch, Suresnes, and INSERM U 24, HSpital Beaujon, Clichy, France. Plasma catecholamines are usually modified in patients with portal hypertension due to cirrhosis but little is known about the actual mechanism of the increased sympathetic activity in portal hypertension. The aim of this study was to compare free and conjugated plasma catecbolamines from arterial and portal venous territories between i0 rats with portal hypertension due to portal vein stenosis and i0 control animals. Blood samples were withdrawn simultaneously in fasted anaesthetized rats from the carotid artery and portal vein. Free, sulfo- and glucurono-conjugated catecholamines were measured in the plasma with the radioenzymatic assay method. In arterial territory, no significant difference in free and conjugated catecholamines was observed between the 2 groups of rats except for free adrenaline which was significantly lower in portal hypertensive rats than in controls (54±7 and 126±3 pg/ml, respectively). Differences in free and conjugated catecholamines between the 2 territories are shown in the table for the 2 groups (portal venous-arterial concentrations expressed in pg/ml). Dopamine Noradrenaline Adrenaline free sulfo glucurono free sulfo glucurono free sulfo glucurono Controls 48 c 1970 c 3575 b 143 c 1235 b -424 b 89 c 976 b -453 a Portai hypertension -4 d 594 a 1990 b 167 c 378 a 530 d -29 c 969 b 1671 c a p < 0.05; b p < 0.01; c p < 0.001; d NS. In conclusion, these results show that in this model of portal hypertension in the rat with normal liver, free arterial plasma adrenaline and glucurono-conjugated portal venous plasma noradrenaline and adrenaline differ from controls. This study suggests that glucurono-conjugation of catecholamines may be deranged in portal hypertension. IMMUNOGOLDLOCALIZATION OF PROCOLLAGENI l l , FIBRONECTINAND HEPARAN SULPHATE PROTEOGLYCANON ULTRATHINFROZEN SECTIONSOF RAT LIVER. 120 A. Geerts °, P. Schellinck °, B. Voss °°, D. Schuppan °a°, E. Wisse °. VLab'. voor Celbiologie en Histologie, Vrije Universiteit Brussel (VUB), Be~gi~ °°Institut fdr Arterioscleroseforschung, Universit~t ~nster, BRD °°°Universit~tsklinikum Steglitz, Freie Universit~t Berlin, BRD Despite numerous studies on the characterization and localization of different elements of the liver extracellular matrix, several aspects in this field remain controversial. It has not yet been established (i) whether collagen type Ill is restricted to fine reticular fibrils, (ii) whether type IV collagen, fibronectin and heparan sulphate proteoglycan contribute to basal lamina like fragments in the space of Disse, and ( i i i ) to what extent parenchymal (PCs) or sinusoidal cells synthesize or degrade connective tissue in normal or diseased liver. In the present study we investigated these questions by means of the protein A-gold tech- nique on ultrathin frozen sections of rat liver. Using antibodies against rat procollagen type III (PIIIP), it was shown that all collagen fibrils in the space of Disse contain PIIIP. By measurement of the diameter of the collagen fibrils in the space of Disse, it was found that they consist of one population of fibrils. These results make it unlikely that the liver con- tains two separate types of fibrils, i.e. "thin" reticular fibrils of type III collagen and "thick" fibrils of type I collagen. With the anti PIIIP, intracellular staining in several PCs and endothelial cells (ECs) was observed. Thus, these cells actively synthesize PIIIP in the normal rat liver. Using antifibronectin and antiheparan sulphate proteoglycan antibodies, we demonstrated the presence of these molecules in the space of Disse in two types of material : (i) in strands, interconnecting PC microvilli with ECs and (ii) in diffuse material closely associated with the membranes of the PCs and ECs. These observations indicate that both fibronectin and hepa- ran sulphate proteoglycan, act as attachment proteins in the space of Disse. $238

Immunogold localization of procollagen III, fibronectin and heparan sulphate proteoglycan on ultrathin frozen sections of rat liver

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Page 1: Immunogold localization of procollagen III, fibronectin and heparan sulphate proteoglycan on ultrathin frozen sections of rat liver

119

FREE AND CONJUGATED PLASMA CATECHOLAMINES IN NORMAL RATS AND IN RATS WITH PORTAL HYPERTENSION DUE TO PORTAL VEIN STENOSIS C. Gaudin, G. Ru~et, F. Selz, C. Girod, JL. Cuche, D. Lebrec INSERM U 27, NSpital Foch, Suresnes, and INSERM U 24, HSpital Beaujon, Clichy, France.

Plasma catecholamines are usually modified in patients with portal hypertension due to cirrhosis but little is known about the actual mechanism of the increased sympathetic activity in portal hypertension. The aim of this study was to compare free and conjugated plasma catecbolamines from arterial and portal venous territories between i0 rats with portal hypertension due to portal vein stenosis and i0 control animals. Blood samples were withdrawn simultaneously in fasted anaesthetized rats from the carotid artery and portal vein. Free, sulfo- and glucurono-conjugated catecholamines were measured in the plasma with the radioenzymatic assay method.

In arterial territory, no significant difference in free and conjugated catecholamines was observed between the 2 groups of rats except for free adrenaline which was significantly lower in portal hypertensive rats than in controls (54±7 and 126±3 pg/ml, respectively). Differences in free and conjugated catecholamines between the 2 territories are shown in the table for the 2 groups (portal venous-arterial concentrations expressed in pg/ml).

Dopamine Noradrenaline Adrenaline free sulfo glucurono free sulfo glucurono free sulfo glucurono

Controls 48 c 1970 c 3575 b 143 c 1235 b -424 b 89 c 976 b -453 a Portai hypertension -4 d 594 a 1990 b 167 c 378 a 530 d -29 c 969 b 1671 c a p < 0.05; b p < 0.01; c p < 0.001; d NS.

In conclusion, these results show that in this model of portal hypertension in the rat with normal liver, free arterial plasma adrenaline and glucurono-conjugated portal venous plasma noradrenaline and adrenaline differ from controls. This study suggests that glucurono-conjugation of catecholamines may be deranged in portal hypertension.

IMMUNOGOLD LOCALIZATION OF PROCOLLAGEN I l l , FIBRONECTIN AND HEPARAN SULPHATE PROTEOGLYCAN ON ULTRATHIN FROZEN SECTIONS OF RAT LIVER.

120 A. Geerts °, P. Schellinck °, B. Voss °°, D. Schuppan °a°, E. Wisse °. VLab'. voor Celbiologie en Histologie, Vrije Universiteit Brussel (VUB), Be~gi~ °°Institut fdr Arterioscleroseforschung, Universit~t ~nster, BRD °°°Universit~tsklinikum Steglitz, Freie Universit~t Berlin, BRD

Despite numerous studies on the characterization and localization of different elements of the l iver extracellular matrix, several aspects in this f ield remain controversial. I t has not yet been established (i) whether collagen type I l l is restricted to fine reticular f ib r i l s , ( i i ) whether type IV collagen, fibronectin and heparan sulphate proteoglycan contribute to basal lamina like fragments in the space of Disse, and ( i i i ) to what extent parenchymal (PCs) or sinusoidal cells synthesize or degrade connective tissue in normal or diseased l iver.

In the present study we investigated these questions by means of the protein A-gold tech- nique on ultrathin frozen sections of rat l iver. Using antibodies against rat procollagen type I I I (PIIIP), i t was shown that all collagen f ibr i ls in the space of Disse contain PIIIP. By measurement of the diameter of the collagen f ibr i ls in the space of Disse, i t was found that they consist of one population of f ibr i ls . These results make i t unlikely that the l iver con- tains two separate types of f ibr i ls , i .e. "thin" reticular f ibr i ls of type I I I collagen and "thick" f ibr i ls of type I collagen. With the anti PIIIP, intracellular staining in several PCs and endothelial cells (ECs) was observed. Thus, these cells actively synthesize PIIIP in the normal rat l iver.

Using antifibronectin and antiheparan sulphate proteoglycan antibodies, we demonstrated the presence of these molecules in the space of Disse in two types of material : (i) in strands, interconnecting PC microvilli with ECs and ( i i ) in diffuse material closely associated with the membranes of the PCs and ECs. These observations indicate that both fibronectin and hepa- ran sulphate proteoglycan, act as attachment proteins in the space of Disse.

$238