Randi Holme

A study of the release of calcium from human blood platelets and its inhibition by metabolic inhibitors, N-ethylmaleimide and aspirin

Abstract 1. 1. The major part of the calcium stored in human blood platelets was released by incubation with thrombin, latex particles or NaF. The release was inhibited by the combined effect of inhibitors of glycolysis and of oxidative phosphorylation, and by N -ethylmaleimide and aspirin. A correlation between the effect of aspirin on release and on platelet metabolism has been demonstrated. 2. 2. The major part of the calcium release took place within the first minute after addition of thrombin or latex particles; after the addition of F − , however, significant release began after 5 min and reached a maximum only after 20 min. 3. 3. Calcium released by thrombin, as well as the calcium remaining after release, was retained after washing with saline, while 45 Ca incorporated into washed platelets in the presence or absence of thrombin could be almost completely removed by washing. This indicates that the released calcium is stored in a form inaccessible to exchange with the extracellular medium. 4. 4. The ratio of calcium to adenine nucleotides in the supernatant was approx. 1.8 while the amount of calcium in platelets averaged about 50 nmoles/mg of platelet protein, or half to one third of that reported by other investigators.

Plasma cholesterol esterification and plasma lipoproteins in bile-duct-ligated dogs.

To study the lipoprotein changes in cholestasis while the capacity for plasma cholesterol esterification was normal, the common bile duct was ligated in dogs and plasma investigated 8 h and 48 h later. The plasma concentration of cholesteryl esters was slightly increased, concomitant with a tendency toward an increase in the activity of lecithin:cholesterol acyltransferase (LCAT). The content of cholesteryl esters in the main lipoprotein classes was normal. Marked alteration in the low density lipoproteins (LDL) and the high density lipoproteins (HDL) took place and were essentially similar 8 h and 48 h after bile duct ligation. In LDL, (density 1.006--1.019 g/ml) and LDL2 (density 1.019--1.063 g/ml) an increase in the content of polar lipids was observed, and in LDL2 heterogeneity in particle size was demonstrated by gelfiltration on 2% agarose and by electron microsopcy. Large myelin structures, flattened disc-shaped particles, and particles with the appearance of normal LDL2 were present. HDL isolated after operation was characterized by a decreased protein/lipid ratio and an increased content of phospholipids. By gelfiltration on Sephadex G-200 and by electron microscopy changes in particle size were observed, with the presence of disc-shaped particles with a tendency in form rouleaux. These results demonstrate that marked lipoprotein changes occur as early as 8 h after bile duct-ligation in dogs and indicate that a deficient LCAT mechanism is present in cholestasis even with normal or high plasma LCAT activity.

Electron microscopy of the gel protein formed by clotting of Limulus polyphemus hemocyte extracts

The Limulus hemocyte extract was clotted by endotoxin, and isolated gel protein was dissolved in buffers of different pH and ionic strength and studied in the electron microscope after negative staining. When the gel protein was dissolved in 0.05 M formate buffer pH 3.0 and pH 4.0, fibers with apparent helical structure were observed. In formate buffer pH 3.0 at different ionic strengths, the tendency of the fibers to exist as aggregates increased when the salt concentration was increased. The fibers were also present as aggregates in 0.05 M carbonate/bicarbonate buffer pH 10.5. Helical substructures were not observed in 0.188 N NaOH. The results indicate that the helical structure of the gel protein is stable over a wide range of pH and ionic strength.

Intercellular adhesion molecule-1 (ICAM-1; CD54) expression in human hepatocytic cells depends on protein kinase C

BACKGROUND/AIMS Intracellular regulation of intercellular adhesion molecule-1 has mainly been studied in lymphoid, endothelial, and epithelial cells. Intercellular adhesion molecule-1 plays a central role in many immune responses, and we have previously studied its regulation in hepatocytes. Here we report how manipulation of intracellular signal systems influenced its expression. METHODS The constitutive and cytokine-induced expression of intercellular adhesion molecule-1 mRNA and protein was studied in the human hepatocytic cell lines Hep G2 and SK-Hep-1. RESULTS When agonists and antagonists of protein kinase C, calmodulin, and protein kinase A were introduced in addition to prostaglandin E2 and a cyclooxygenase inhibitor, only the protein kinase C activator phorbol 12-myristate 13-acetate resulted in a rapid and dose-dependent increase in intercellular adhesion molecule-1 protein and mRNA. Phorbol 12-myristate 13-acetate stimulated sustained high levels of intercellular adhesion molecule-1 protein, whereas the corresponding mRNA response was biphasic, peaking at 3 h. Actinomycin D blocked the stimulatory mRNA phase, suggesting that de novo transcription was induced. Coincubation with phorbol 12-myristate 13-acetate and the protein synthesis inhibitor cycloheximide gave considerably higher mRNA levels than with phorbol 12-myristate 13-acetate alone. Protein kinase C may therefore even stimulate synthesis of proteins that speed up the turnover of intercellular adhesion molecule-1 mRNA. The protein kinase C inhibitor staurosporine abrogated the induction of intercellular adhesion molecule-1 by phorbol 12-myristate 13-acetate, indicating that this effect was indeed exerted by protein kinase C. More original was our observation that staurosporine also completely blocked the stimulatory effects of interferon-gamma, tumour necrosis factor-alpha, and interleukin-1. Recent reports have noted that these cytokines apparently use receptors which activate different intracellular pathways. We also noted that the glucocorticoid dexamethasone partially inhibited the stimulation of intercellular adhesion molecule-1 by these cytokines. This phenomenon could be important for the immunosuppressive effects of corticosteroids in patients with liver disease. CONCLUSIONS Our data suggest that a certain level of protein kinase C activity is mandatory for liver cells in cytokine-mediated upregulation of intercellular adhesion molecule-1.

The charge-heterogeneity of human fibrinogen as investigated by 2D electrophoresis

The charge-heterogeneity of human plasma fibrinogen subunit chains was characterized by two-dimensional electrophoresis (2DE). Western blotting with antibodies specific for the gamma-chain demonstrated that the gamma-chains focus at varying isoelectric points (pI). This microheterogeneity was also observed in fibrinogen secreted from hepatocytic cells and in recombinant fibrinogen expressed in Chinese hamster ovary (CHO) cells. Further, covalent gammagamma-dimerization by FXIIIa was not influenced by the charge-heterogeneity, and removal of the carbohydrate did not reduce the number of gamma-chain pI variants. These observations suggest that the microheterogeneity of the gamma-chain is a multifactorial phenomenon that is not due to physiologic modification of the glycoprotein in circulation.

Familial lecithin:cholesterol acyltransferase deficiency. Further studies on plasma lipoproteins and plasma postheparin lipase activity of a patient with normal renal function.

Plasma lipoproteins and postheparin plasma were investigated in a patient with familial LCAT deficiency with normal renal function and without proteinuria. As revealed by gelfiltration the large molecular weight LDL2 was not present, and myelin structures were not found in LDL1 or LDL2 when she was on her ordinary diet. After 60--65% fat diet for one week the large molecular LDL2 was found, but only in low concentration. We have no explanation for the difference in the lipoprotein abnormalities of this patient and others with this disease. There is no major difference in the fat content in the ordinary diet of the Norwegian patients with familial LCAT deficiency, nor has our patient any clinical signs of malabsorption. Furthermore, there was no difference in lipoprotein lipase or hepatic lipase activity in postheparin plasma between our patient and others with the same disease. However, whereas hepatic lipase activity was within the reference values, lipoprotein lipase activity was rather low in all patients investigated. We suggest that impaired VLDL catabolism in plasma, because of LCAT deficiency and low lipoprotein lipase activity, may partly explain the low HDL concentration consistently found in patients with familial LCAT deficiency.

Triglyceride lipase activity in postheparin plasma and plasma lipoproteins in liver disease

Hepatic lipase activity and lipoprotein lipase activity were studied in postheparin plasma from 14 patients with various liver disorders. Plasma lecithin: cholesterol acyltransferase (LCAT) activity and lipoprotein composition and structure were also estimated. Five patients had lower hepatic lipase activity than the lowest control value, and in three of these no hepatic lipase activity was detected. Lipoprotein lipase was low in 5 patients, but in only one of them was hepatic lipase activity also low. Hepatic lipase was not significantly correlated to the concentration of plasma triglycerides, either in controls or in patients, whereas lipoprotein lipase was negatively correlated with plasma triglycerides both in controls and patients. Lipoprotein lipase and LCAT activity, but not hepatic lipase, was negatively correlated to the triglyceride content of the low density lipoproteins (density 1.019-1.063 g/ml) from the patients. No specific lipid or lipoprotein pattern was found in plasma from the patients with a low or without any hepatic lipase activity. The results suggest an important role of lipoprotein lipase and LCAT, for the increased content of triglycerides in the low density lipoproteins in patients with liver disease. The role of hepatic lipase remains unclear.

Lipid deposition in kidneys in experimental liver disease: a study in dogs with choledochocaval anastomosis.

Abstract. To study the ultrastructural changes in kidneys in an experimental model with an increase in the plasma concentration of lipoproteins rich in free cholesterol and phospholipids, cholecystectomy and choledochocaval anastomosis were performed in four dogs. In three dogs the glomeruli were markedly abnormal with deposition of osmiophilic material mainly in the subendothelial regions. The isolated glomeruli contained increased amounts of free cholesterol and phospholipids. In these dogs the plasma concentrations of free cholesterol and phospholipids rose markedly and cholesteryl esters fell. Abnormal plasma lipoprotein particles ultrastructurally similar to those seen in human cholestatic liver disease and in familial deficiency of the enzyme lecithin:cholesterol acyltransferase (LCAT) were found. Large myelin structures in the low density lipoprotein (LDL) range were especially prominent. Serum creatinine and urea concentrations were normal.

The electrophoretic mobility of lipoprotein x in postheparin plasma

After incubation whole plasma and low density lipoproteins (LDL) taken before and 10 min after intravenous administration of heparin, from a patient with primary biliary cirrhosis and a patient with familial lecithin:cholesterol acyltransferase (LCAT) deficiency, have been tested for the presence of lipoprotein X (LP-X) by agar gel electrophoresis. LP-X was present in preheparin whole plasma and LDL. No precipitation lines on the cathodal side of the wells, indicating the absence of LP-X, were seen after electrophoresis of postheparin plasma and LDL. Immunodiffusion revealed the presence of apo-beta-lipoproteins and LP-X in preheparin as well as postheparin LDL. After gel filtration three subfractions and similar patterns were observed in the preheparin and postheparin LDL. Electronmicroscopical examination of the intermediate subfractions showed LP-X-like particles in preheparin and postheparin samples. These observations indicate a changed electrophoretic mobility in agar gel of postheparin LP-X, giving a false negative LP-X test by the conventional agar gel electrophoresis.

The effect of lipoprotein lipase and hepatic lipase on the electrophoretic mobility of lipoprotein-X

Lipoprotein-X containing plasma from a patient with familial lecithin:cholesterol acyltransferase (LCAT) deficiency, was used as substrate and incubated with postheparin plasma or partly purified lipases. LP-X could not be demonstrated by agar gel electrophoresis after incubation with postheparin plasma from a healthy subject, from a patient with chronic active hepatitis deficient in hepatic lipase, or with partly purified lipoprotein lipase. After incubation a marked increase in free fatty acids (FFA) was observed. In contrast LP-X was still present after incubation when postheparin plasma deficient in lipoprotein lipase or partly purified hepatic lipase was added to the substrate. Only minor changes in the concentration of FFA occurred. After addition of oleic acid to the substrate LP-X could not be demonstrated by agar gel electrophoresis. However, in the isolated low density lipoproteins, LP-X like particles were still present as viewed by electron microscopy. Our results strongly suggest that the change in electrophoretic mobility of LP-X was induced by the release of FFA. This was achieved by lipoprotein lipase, but not by hepatic lipase.