Role of nitric oxide increase on induced programmed cell death during early stages of rat liver regeneration

We analysed the possible cellular mechanism involved in the NO action in the balance between apoptosis and cell proliferation in liver regeneration process. We determined p53, proapoptotic protein Bax, antiapoptotic Bcl-xL, proliferating cell nuclear antigen (PCNA) and apoptotic index at the early stages of regenerative process after NO increase by lipopolysaccharide-induction (LPS) of inducible-type nitric oxide synthase (iNOS) and by direct NO donor (sodium nitroprusside, SNP). Male Wistar rats were randomised in four experimental groups: sham operated control (Sh), partial hepatectomised control (PH-C), partial hepatectomised pretreated with LPS (2 mg/kg body weight, i.p.) (PH-LPS), and partial hepatectomised pretreated with SNP (2.5 mg/kg body weight, i.v. at a rate of 1 ml/h) (PH-SNP). Animals were killed 5 h post-surgery. Hepatic cytosolic iNOS showed an increase of 34% in PH-C animals with respect to Sh, and LPS-treatment increased iNOS protein levels 30% compared with PH-C. Bax and p53 protein levels showed significant increases in LPS- and SNP-treated hepatectomised rats with respect to PH-C. The apoptotic indexes were increased 75% in both, PH-LPS and PH-SNP rats versus PH-C. The increase of NO did not show any change in the proliferation process. These results suggest that NO is involved in apoptosis via p53 and Bax proteins after PH, showing a tightly regulated growth process in liver regeneration.     

Post-heparin plasma lipolytic activity in acute hepatitis

Post-heparin plasma lipolytic activity (PHLA) was measured in 21 patients with acute viral hepatitis. PHLA was significantly reduced in all patients (p less than 0.0005), whose mean activity was only 32% of the control subjects. An inverse correlation (p less than 0.05) was found between PHLA values and serum triglycerides levels. During recovery, the normalization of PHLA was much slower than that of the usual tests of liver function. PHLA measurement may represent a new and sensitive test of liver function.     

Glucagon action upon plasma post-heparin lipolytic and monoglyceride hydrolase activities. Studies involving administration of exogenous hormone during suppression of endogenous hormone secretion with somatostatin

It has been previously demonstrated that glucagon increased plasma post-heparin lipolytic activity (PHLA) in normal rats, but this was not the case in alloxan diabetic rats. The present work was designed to determine if the administration of exogenous glucagon (0.2 mg i.v.) during suppression of endogenous hormone secretion with somatostatin modifies the plasma post-heparin lipolytic activity in normal rats and the action of such hormone upon monoglyceride hydrolase (MGH) activity. It was found that exogenous glucagon significatively increased PHLA and MGH activity in normal rats after 18-24 hours of starvation. However, both enzymatic activities were not influenced by exogenous glucagon when they were measured during somatostatin administration. Therefore it is believed that the enhancement of these activities observed when somatostatin was not simultaneously given was due to the insulin secretion that follows the glucagon injection.     

Effect of dietary carbohydrate type on lipoprotein lipase, hepatic lipase, and lecithin:cholesterol acyltransferase activities in cynomolgus monkeys

The effects of dietary sucrose and starch with and without exogenous cholesterol on postheparin plasma lipoprotein lipase (PHLA) and hepatic lipase (HLA) were studied in cynomolgus monkeys. Serum triglyceride levels were higher in sucrose-fed animals than starch and exogenous cholesterol lowered serum triglyceride levels when added to sucrose diet but not starch diets. Sucrose markedly increased insulin levels, more so than starch; however, dietary cholesterol lowered insulin levels in sucrose diet but increased the levels in starch diet. PHLA activity was increased two- to threefold greater in sucrose than in starch diets. Exogenous cholesterol lowered PHLA activity in sucrose diet but increased PHLA activity in starch diet. HLA activity was increased with sucrose more than starch. Lecithin:cholesterol acyltransferase (LCAT) activity was significantly higher in sucrose diets than in the starch diet. Addition of cholesterol to either of these diets lowered the LCAT activity. These results indicate that PHLA, HLA, and LCAT activities not only are affected by the nature of carbohydrates, but also are related to triglyceride metabolism. The interaction of carbohydrates and cholesterol in the diet by influencing these selected enzymes plays an integrated role in lipoprotein particle interconversion processes.     

Proteinsynthese in isolierten Leberzellkernen nach partieller hepatektomie

Incubation of liver nuclei from hepatectomised rats with radioactive amino acids shows that the rates of synthesis of the globular and residual protein fractions increase linearly for up to 24 hr after hepatectomy, whereas the histones show maximal incorporation at 15 hr.     

Lipid peroxidation in regenerating rat liver

Rats entrained to a strictly regulated lighting and feeding schedule have been subjected to partial hepatectomy or a sham operation. In the partially hepatectomised animals the period of liver regeneration is characterised by regular bursts of thymidine kinase activity. Liver microsomes from rats, at times corresponding to maximum thymidine kinase activity, have much reduced rates of lipid peroxidation compared to control preparations: this is due in part to increased levels of lipid-soluble antioxidant at times of maximal DNA synthesis. This temporal relationship between thymidine kinase and lipid peroxidation is consistent with the view that lipid peroxidation is decreased prior to cell division.     

Effects of ingestion of thermally oxidized edible oils on plasma lipids, lipoproteins and postheparin lipolytic activity of rats

Acute (after 4 hr) and short-term (after 7 days) effects of ingesting heated and unheated groundnut, coconut and safflower oils on plasma lipids, lipoproteins and postheparin lipopolytic activity (PHLA) were studied in rats. All heated oils were characterized by increases in carbonyl value, peroxide value and free fatty acid (FFA) content, except heated coconut oil which showed a decrease in FFA content. Heating procedure also did not alter to an appreciable extent their fatty acid compositions. Acute and short-term effects of feeding heated and unheated oils showed no significant differences in rat plasma levels of total cholesterol (TC), total triglycerides, total phospholipids, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol + very-low-density lipoprotein cholesterol, TC/HDL-C ratio and PHLA. Inspite of certain changes in some of the indices of thermal alteration of these heated oils, consumption of such heated oils by rats did not have any significant effect on various plasma parameters in these animals.     

Effects of diabetes on glucose oxidation in rat aorta after hypophysectomy and adrenalectomy

The influence of diabetes, hypophysectomy and adrenalectomy on glucose oxidation in rat aorta was studied. Diabetes was induced in normal, adrenalectomized and hypophysectomized-cortisone substituted rats by streptozotocin (65 mg/kg body weight). The oxidation of glucose to CO2 was determined during incubation of rat aorta in vitro for 2-3 hours. The aortic glucose oxidation was reduced after hypophysectomy but was unaffected by adrenalectomy. After streptozotocin treatment the rise in blood glucose concentration was similar in normal, adrenalectomized and hypophysectomized-cortisone substituted rats. In shamoperated diabetic rats the aortic glucose oxidation was reduced after a diabetes duration of 4 days. In adrenalectomized diabetic rats the aortic glucose oxidation was not significantly affected after 4 days but was reduced after a diabetes duration of 14 days. When adrenalectomized diabetic rats were treated with hydrocortisone the aortic glucose oxidation was reduced after diabetes for 4 days. After incubation of normal rat aorta in vitro for 6 hours with cortisol (1 microgram/ml) in the incubation medium a decrease in the aortic glucose oxidation was found. Incubation of aorta with only growth hormone had no effect. These results suggest that cortisol is of importance for the lowered glucose oxidation in diabetic rat aorta.     

Brain Tryptophan Hydroxylation in the Portacaval Shunted Rat: A Hypothesis for the Regulation of Serotonin Turnover In Vivo

Regional and whole-brain tryptophan-hydroxylating activity and serotonin turnover were investigated in portacaval shunted (PCS) rats using an in vivo decarboxylase inhibition assay. To saturate tryptophan hydroxylation with amino acid substrate, rats were administered a high dose of tryptophan 1 h prior to analysis of brain tryptophan, 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid. The analysis revealed, as expected, higher brain concentrations of tryptophan and 5-hydroxyindoles and increased serotonin synthesis rate in PCS rats as compared with shamoperated controls. Saturating levels of brain tryptophan were achieved in both PCS and sham animals after exogenous tryptophan administration. The tryptophan load resulted in increased brain serotonin turnover in all regions and in whole brain compared with rats that did not receive a tryptophan load. Tryptophan-loaded PCS rats showed increased brain serotonin turnover compared with tryptophan-loaded sham rats. Regionally, this supranormal tryptophan-hydroxylating activity was most pronounced in the mesencephalon-pons followed by the cortex. It is concluded that, at least in the PCS rat, brain tryptophan hydroxylation is an inducible process. Since it is known that brain tissue from PCS rats undergoes a redox shift toward a reduced state and that the essential cofactor tetrahydrobiopterin is active in tryptophan hydroxylation only when present in its reduced form, it is hypothesized that this is the reason for the supranormal tryptophan-hydroxylating activity displayed by the PCS rats. The hypothesis further suggests that alterations in tetrahydrobiopterin availability may serve as a mechanism by which brain tryptophan hydroxylation, and therefore serotonin turnover, can be regulated with high sensitivity in vivo.     

The interaction of ( 3 H)ethyl carbamate with nucleic acids of regenerating mouse liver

The binding of [³H]urethane to liver DNA and RNA has been examined in partially hepatectomised and intact male Crackenbush mice. A single dose of [³H]urethane (50 μCi) was given to non-hepatectomised mice (group A) and to 3 groups of partially hepatectomised mice at 18 (B), 28 (C) and 38 (D) hours postoperatively, respectively. The binding was examined over the subsequent 16 h. The maximum levels of binding to DNA declined in the order, group A > B > C > D, although the binding to DNA persisted longest in group B. The binding to RNA was greater in groups B, C and D than in group A. Neither the restoration of liver mass nor an alteration in the metabolism of urethane appeared to account for the different levels of binding. In normal and partially hepatectomised mice a single dose of urethane (20 mg) was followed by an inhibition of mitosis and of the incorporation of [³H]thymidine into liver DNA and of [³H]uridine into liver RNA.     

Effects of oxytocin and vasopressin on retrieval of passive avoidance response in melatonin-treated and/or pinealectomized male rats

The role of the pineal gland and its hormone-melatonin-as to the impact of vasopressin (VP) and/or oxytocin (OT) on the regulation of behavior was studied, the passive avoidance task being chosen as an experimental model. The results showed that VP facilitated the avoidance latency during the first retention trial; after pinealectomy, however, VP was ineffective in this regard. Intraperitoneal application of OT was ineffective in modifying the passive avoidance latency when compared with respective saline-treated animals. Melatonin alone, when injected to shamoperated animals 30 min before behavioral experiment, did not affect the passive avoidance response in SA- or OT-treated rats, but blocked the VP-induced lengthening of the passive avoidance latency in the first retention trial. In pinealectomized and OT-treated animals the passive avoidance latency during the second retention trial was severely diminished by melatonin when compared to respective control. We conclude that: a) VP needs a regulated pineal function for developing short-term effects on the passive avoidance response and b) the effect of OT on the avoidance latency in pinealectomized rats develops after melatonin treatment as a long-term effect.     

Effect of a single treatment with the alkylating carcinogens dimethynitrosamine, diethylnitrosamine and methyl methanesulphonate, on liver regenerating after partial hepatectomy. I. Test for induction of liver carcinomas.

A single injection of dimethylnitrosamine (DMN), 12.0-15.6 mg-kg, given to 100 g female rats 24 h after partial hepatectomy, induced hepatocellular carcinoma. No animals receiving DMN without partial hepatectomy developed liver carcinomas. Previous evidence had suggested that the incidence of tumours was highest when DMN was administered during the wave of DNA replication which follows partial hepatectomy. The present experiments made this suggestive evidence statistically significant. A single treatment with diethylnitrosamine (DEN) induced liver cell cancer when given to intact or to partially hepatectomised rats. No tumors developed when another alkylating carcinogen, methyl methanesulphonate (MMS), was administered after partial hepatectomy. The significance of these results in relation to the mechanism of initiation of carcinogenesis is discussed.     

High fructose intake significantly reduces kidney copper concentrations in diabetic, islet transplanted rats

Trace element status is known to be altered in the diabetic state, although the factors affecting trace element homeostasis in this condition are not well understood. The authors examined the effects of a high fructose diet (40% wt:wt) vs a control diet on the copper (Cu), zinc (Zn), and iron (Fe) concentrations in the kidney, plasma, and red blood cells of islet transplanted (TX) and shamoperated (SHAM) rats. Male, Wistar Furth rats made diabetic by streptozotocin injection (55 mg/kg, iv) were given an intraportal islet transplant (1000 islets); control animals were shaminjected, shamoperated (SHAM). Rats within TX and SHAM groups were assigned to either a high fructose diet (40% fructose, 25% cornstarch, FR) or a purified control diet (33% cornstarch, 33% dextrose, CNTL) containing identical amounts of mineral mixture for a period of 6 wk. Kidney Cu concentration was significantly elevated among hyperglycemie TXCNTL rats (224 ± 25 nmol/g wet wt), but was markedly reduced in hyperglycemic TXFR rats (109 ± 14 nmol/g) relative to normoglycemic controls. This occurred in spite of similar levels of glucose, insulin (fed and fasted), insulin secretory capacity, body weight, and food intake in the TXCNTL and TXFR groups. Among the subgroup of rats with normal glucose levels post-TX, kidney Cu levels normalized and were unaffected by dietary treatment (normoglycemic TXCNTL = 60 ± 5 nmol/g; normoglycemic TXFR = 40 ± 2 nmol/g). Kidney Cu concentrations also were unaffected by fructose feeding in SHAM animals (CNTL, 60 ± 4 nmol/g and FR, 51 ± 5 nmol/g). Kidney Zn and Fe concentrations were similar among the treatment groups. Plasma and red blood cell (RBC) Cu, Zn, and Fe concentrations were also similar among the groups. Since fructose feeding led to a substantial reduction of kidney Cu concentrations in the presence of hyperglycemia, the authors suggest that this model can be useful in examining effects of altered kidney Cu accumulation in the diabetic animal.     

Fetal hypothalamic transplants in the third ventricle of the adult rat brain

Summary Blocks of anterior hypothalamus were transplanted from 19 day-old fetuses of Wistar/Lewis rats into the third ventricle of adult male Brattleboro rats. Physiological changes in graft recipients and in sham-operated animals were monitored daily. Twenty days after surgery, the graft recipients and shamoperated animals were killed and their brains examined by correlative scanning and transmission electron microscopy. Host animals that exhibited both decreased polydipsia and increased urine concentration were found to have viable grafts within the third ventricle. The observed physiological changes suggested that synthesis and release of vasopressin occurred in the transplanted neurons. Grafts were well vascularized by vessels arising from the host hypothalamus. Neurons, with perikarya ranging from 8 to 30 m in diameter, glial cells, and neurites were located throughout the transplants. A neurohemal contact zone, similar to that normally seen in the median eminence, could not be demonstrated in the grafts. The absence of complete glial and ependymal barriers indicates a relatively close association between cells in the transplants and the cerebrospinal fluid. A large increase in supraependymal neurons and their processes, including an eruption of neurons through the floor of the third ventricle in one animal, was observed in graft recipients but not in shamoperated animals.Supported by NIH Grants NS 15109 and NS 13717     

Effect of aflatoxin B1 on phosphoinositide signal transduction pathway during regeneration of liver cells following partial hepatectomy.

Aflatoxin B1 (AFB1) when administered to partially hepatectomised rats 4 hr prior to sacrifice, activated the signalling pathway in regenerating rat liver. The activity of phosphatidylinositol (PI) kinase was found decreased at 30 min but increased at 24 hr and returned to normal at 48 hr. At 30 min, inositol-1,4,5-triphosphate (IP3) level increased significantly whereas diacylglycerol (DAG) level dropped. However, at 24 hr and 48 hr, DAG and IP3 showed the same trend i.e. an increase in their levels. Phosphatidylinositol-4-phosphate levels were found to increase at 24 hr. Protein kinase C (PKC), activity from the particulate fraction was significantly inhibited at 30 min, followed by increase in activity at 24 hr and return to normal at 48 hr. Cytosolic PKC showed a decrease at 24 hr and a significant increase at 48 hr. At the peak of DNA synthesis (24 hr) following partial hepatectomy, all these signalling steps had earlier been found to be inhibited, but the present study shows that aflatoxin B1 administration 4 hr prior to sacrifice reverses the action. Activation of PKC by aflatoxin B1, during regeneration of liver cells when PKC in normally inhibited, may possibly create conditions conducive to carcinogenesis.     

The lipoprotein lipase system: new understandings.

Hypertriglyceridemia, a risk factor for premature atherosclerosis, may result from decreased use of plasma triglycerides by tissues. The removal of triglycerides is mediated by the enzyme lipoprotein lipase (LPL). Heparin releases LPL from tissues and post-heparin plasma lipolytic activity (PHLA) has been extensively used to elucidate the mechanism of hypertriglyceridemia in various diseases. There is evidence to show that postheparin plasma contains enzymes other than LPL. Hence data on total PHLA are difficult to interpret. Availability of assays for the LPL component of PHLA has clarified equivocal findings in certain hypertriglyceridemic states. However, the LPL component is also heterogeneous. The LPL "isoenzymes" from various extrahepatic tissues behave differently under various metabolic conditions. Therefore, to understand properly the LPL system it is necessary to study the specific tissue LPL. Furthermore, the serum activator for LPL is now characterized. Its importance is evidenced by the recent discovery of a hypertriglyceridemic patient deficient in this apoprotein.     

Effect of partial hepatectomy on the invertor mechanism of regulation of the Na+,K+-ATPase activity in hepatocytes of rats of various ages]

Age peculiarities of partial hepatectomy effect on the hepatocytes plasma membrane Na+, K(+)-ATPase activity and its insulin-induced stimulation has been studied. It has been shown that partial hepatectomy does not change basal Na+, K(+)-ATPase activity in adult rats. In old partial hepatectomised rats Na+, K(+)-ATPase activity is slightly higher than in control old rats, although this increase is not statistically significant. At the same time, partial hepatectomy acts differently on the insulin-induced Na+, K(+)-ATPase activation in adult and old rats. Insulin activates Na+, K(+)-ATPase at the same extent both in control and partial hepatectomized adult animals. In old hepatectomized rats, but not in old control animals, insulin stimulates Na+, K(+)-ATPase activity as well as. Thus hepatectomy "rejuvenates" old hepatocytes and results in recovery of invertor mechanism of Na+, K(+)-ATPase activation.     

Degradation and conversion of somatostatin in normal and diabetic rats in vivo and in vitro

Plasma somatostatin-like immunoreactivity in the portal and jugular veins of streptozotocin diabetic rats was compared with that in normal control rats. In the diabetic group, somatostatin levels in the portal (p less than 0.05) and jugular (p less than 0.01) veins were both elevated compared with those in the control group. Moreover, the degree of elevation was greater in the jugular vein than in the portal vein. To further investigate the role of the liver in the clearance of somatostatin-28 in vivo, 2 micrograms of somatostatin-28 was administered as a bolus into the external jugular vein of intact and functionally hepatectomized rats. The mean half-time of somatostatin-28 was significantly longer in intact diabetic rats than in controls (p less than 0.05). The functional hepatectomy did not cause a significant difference in the half-time in diabetic rats but made it longer in control rats. These results suggest that the longer half-time of somatostatin-28 in diabetic rats in vivo is due to its slower hepatic clearance. The hepatic clearance of somatostatin-28 and somatostatin-14 was further studied in vitro using a recirculating liver perfusion method. The hepatic clearance of 1.2 nM of either somatostatin-28 or somatostatin-14 was significantly lower in diabetic rats than in controls (p less than 0.01). This indicates that elevated plasma somatostatin levels in diabetic rats are caused at least in part by decreased hepatic clearance of somatostatin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of covalently bound heparin coating on patency and biocompatibility of long-term indwelling catheters in the rat jugular vein

Many physiologic and pharmacologic studies require long-term vascular access for repeated substance infusion and/or blood sample collection. The study reported here was undertaken to determine whether a functionally active heparin coating would improve long-term patency of venous catheters in rats. Uncoated or coated catheters were surgically placed in the jugular vein, and patency was evaluated twice weekly for a total of 30 days. Culturing of blood and catheters, and histologic examination were performed for all rats. All heparin-coated catheters remained patent for the study duration, with patency defined as ability to infuse saline and withdraw a blood sample. Median patency for uncoated catheters was 17.5 days, with a range of three to 30 days. Histologic evaluation of vessels revealed more advanced and severe lesions in rats with uncoated, compared with coated catheters. Furthermore, uncoated catheters had increased association with bacteremia (3/8), compared with coated (0/9) catheters. Taken together, these results indicate that coating catheters with covalently bound heparin molecules can significantly prolong patency and cause less pathologic damage to the catheterized vessel.     

A new technique of coronary artery ligation: Experimental myocardial infarction in rats in vivo with reduced mortality

In vivo models of myocardial infarction following coronary artery ligation in the rat still suffer from high early mortality and a low rate of success of myocardial infarction. This study investigated the possibility of reducing early mortality and increasing the rate of myocardial infarction by modifications of surgical techniques. Eighteen rats were divided into two groups: normal control (3 rats) and ligation (15 rats). The major modifications of surgical techniques used in this study include: (1) no exteriorization of the heart, (2) ligation of the origins of the branches rather than the main trunk of the left coronary artery, (3) removal of air from the chest after closure, (4) supplying oxygen immediately after extubation. Following surgery, the rats recovered uneventfully and 11 rats were alive after 16 weeks. One rat, with a large myocardial infarction, died 2 h after surgery. Early mortality (during surgery and 1 week after surgery) was 6.7% with a success rate of myocardial infarction of 85%. The left ventricle in the ligation group showed significant dilation relative to normal and shamoperated control hearts (317% of control hearts, p < 0.001). However, myocardial mass did not increase. The average infarct size was 33%. These results demonstrate that a reduction in early mortality and an increased success rate of myocardial infarction can be achieved by modifications of surgical techniques.