Chieko Mineo

Effect of PGI2 on transcellular transport of fluorescein dextran through an arterial endothelial monolayer

The effects of prostacyclin (PGI2) and stable derivatives of PGI2, such as isocarbacyclin (PGI2 deriv. (A] and isocarbacyclin methyl ester (PGI2 deriv. (B)), on junctional transport of fluorescein dextran (FD) through cultured porcine arterial endothelial cells were investigated. These PGI2S inhibited the transcellular transport dose-dependently. After the elimination of PGI2, its inhibitory effect persisted for at least 1 hr. A good correlation was found between increase of cAMP and the potency of inhibition. Increase of cAMP after PGI2 treatment seemed to be involved in the inhibition of FD transport.

Transcellular transport of angiotensin II through a cultured arterial endothelial monolayer

We have studied the mechanisms of angiotensin II (A-II) transport through a cultured arterial endothelial cell monolayer. The transport of 125I-labeled A-II was inhibited by excess unlabeled A-II (50 microM) and [Sar1, Ile8]-A-II (50 microM), but was not inhibited by bradykinin (50 microM). The transport process was shown to be temperature dependent and was inhibited by 10 mM NaN3 plus 50 mM 2-deoxyglucose. Monensin (50 microM), an inhibitor of endocytotic trafficking, reduced the rate of transport of 125I-A-II. It is also shown that the specific pathway for A-II transport was unidirectional from the apical to the basolateral surface of the endothelial cell monolayer.

Angiotensin II binding activity in cultured porcine arterial endothelial cells.

Angiotensin II (A II) binding activity was detected in the particulate fraction (100,000 g, 60 min precipitate) of cultured porcine aortic endothelial cells. Scatchard analysis of the binding activity indicated a single class of binding sites with a dissociation constant (Kd) of 1.1 nM and a total binding capacity (Bmax) of 125 fmol/mg protein. The binding of [125I]A II was inhibited by excess unlabelled A II, A II analogues ([Sar1, Ile8]A II and [Sar1, Ala8]A II), A I (angiotensin I) and A III (angiotensin III), but not by bradykinin. Type specific A II receptor antagonists, losartan (type 1 angiotensin II receptor) and PD123319 (type 2 angiotensin II receptor), did not inhibit the binding. These results suggest that the A II specific binding protein(s) or receptor(s) is present in arterial endothelial cells, and that it is different from typical type 1 and type 2 angiotensin II receptors.

Intracellular action of an exogenous low-molecular-weight synthetic protease inhibitor, E3123, in cerulein-induced acute pancreatitis in rats

SummaryThe intracellular distribution and action of a new synthetic protease inhibitor, E3123, were studied in cerulein-induced acute pancreatitis in rats. Acute pancreatitis was induced by a 4-h iv infusion of a supramaximal dose of cerulein, and was treated by prophylactic (pretreatment) or therapeutic (posttreatment) continuous administration of E3123. Pancreatic edema and hyperamylasemia were ameriolated only by prophylactic treatment. A subcellular fractionation study showed that the activities of cathepsin-B and trypsin in the zymogen granule-enriched fraction of the cerulein-pancreatitis group were remarkably increased. Both prophylactic and therapeutic treatment significantly prevented the elevation of these enzyme activities. These effects were accompanied by amelioration of pancreatic histopathological features, including intracellular vacuolization and fat necrosis. A microscopic autoradiographic study using2H-labeled E3123 showed diffuse intracellular distribution of E3123, and the radioactivity of2H-E3123 in the posttreatment group was three times greater than that in the pretreatment group. This study provides the first experimental evidence that, even when administered therapeutically, exogenous protease inhibitors are transported into pancreatic acinar cells, thereby reducing the seventy of early intracellular alterations in cerulein-induced acute pancreatitis.

Effect of PGI2 on platelet binding to partially denuded endothelial monolayer in vitro

We have developed a new model for the investigation of platelet interaction with injured vascular endothelium. This involves the quantitative detection of platelet binding to a partially denuded endothelial cell monolayer in vitro. Porcine arterial endothelial monolayer, cultured on collagen gel containing fibrinogen and fibronectin, was partially denuded and the binding of 51Cr-platelets was measured. A synergistic increase in platelet binding was observed in the presence of fibrinogen and fibronectin. A distinct aggregation of platelets along the edge of the denuded area of the endothelial monolayer was seen. Prostacyclin (PGI2) inhibited platelet aggregation, although adhesive platelets were still present at denuded sites.

Exocrine function of caerulein-induced acute pancreatitis in anesthetized rats

Exocrine function was studied in anesthetized rats that had received two specific doses of caerulein (maximal stimulation and supramaximal stimulation). Male Wistar rats (body weight, 200–250 g) were divided into three groups: the control group (4-h saline infusion), the maximal stimulation group (0.25 μg/kg per h caerulein for 4 h), and the caerulein pancreatitis group (10μg/kg per h for 4 h). Histologically, inter-stitial edema and cytoplasmic vacuolization were observed only in the caerulein pancreatitis group, with no abnormal findings in the other groups. The volume of pancreatic juice was significantly increased in both the maximal stimulation group and the caerulein pancreatitis group. The protein ouput and the amylase output in the 1st h of caerulein infusion were also significantly increased, to 459% and 338% in the maximal stimulation group, and to 925% and 1430% respectively, in the caerulein pancreatitis compared to the baseline values. We also found that the pancreatic juice of the caerulein pancreatitis group contained precipitated protein, and high trypsin activity, and protein degradation was confirmed by electrophoresis. These findings were not observed in the other groups. These results strongly suggest that hypersecretion and the appearance of trypsin activity in pancreatic juice plays an important role in the induction of histological changes in this pancreatitis model in anesthetized rats.

Intercellular transport through a partially denuded arterial endothelial monolayer. Effect of platelets and PGI2

Fluorescein Dextran (FD) was shown to be transported at increased rates through partially denuded endothelial monolayer. Platelet binding to the partially denuded monolayer lowered transport rates to those comparable with intact endothelium. Inhibition of transport by platelet binding was not affected by the addition of isocarbacyclin (a stable derivative of PGI2). This result suggests that adherent platelets at the partial denudation site are sufficient to suppress transport of FD.

Endothelial Injury and Accumulation of Cholesterol Ester Derived from Circulating Lipoproteins

To elucidate the mechanism how lipoproteins transport through the endothelial barrier, an in vitro model of transcellular transport of macromolecules was developed.