Robert G. Lamb

Alterations in Phosphatidylcholine Metabolism of Stretch‐Injured Cultured Rat Astrocytes

Abstract: The primary objective of this study was to determine the influence of stretch‐induced cell injury on the metabolism of cellular phosphatidylcholine (PC). Neonatal rat astrocytes were grown to confluency in Silastic‐bottomed tissue culture wells in medium that was usually supplemented with 10 µM unlabeled arachidonate. Cell injury was produced by stretching (5–10 mm) the Silastic membrane with a 50‐ms pulse of compressed air. Stretch‐induced cell injury increased the incorporation of [3H]choline into PC in an incubation time‐ and stretch magnitude‐dependent manner. PC biosynthesis was increased three‐ to fourfold between 1.5 and 4.5 h after injury and returned to control levels by 24 h postinjury. Stretch‐induced cell injury also increased the activity of several enzymes involved in the hydrolysis [phospholipase A2 (EC 3.1.1.4) and C (PLC; EC 3.1.4.3)] and biosynthesis [phosphocholine cytidylyltransferase (PCT; EC 2.7.7.15)] of PC. Stretch‐induced increases in PC biosynthesis and PCT activity correlated well (r = 0.983) and were significantly reduced by pretrating (1 h) the cells with an iron chelator (deferoxamine) or scavengers of reactive oxygen species such as superoxide dismutase and catalase. The stretch‐dependent increase in PC biosynthesis was also reduced by antioxidants (vitamin E, vitamin E succinate, vitamin E phosphate, melatonin, and n‐acetylcysteine). Arachidonate‐enriched cells were more susceptible to stretch‐induced injury because lactate dehydrogenase release and PC biosynthesis were significantly less in non‐arachidonate‐enriched cells. In summary, the data suggest that stretch‐induced cell injury is (a) a result of an increase in the cellular level of hydroxyl radicals produced by an iron‐catalyzed Haber‐Weiss reaction, (b) due in part to the interaction of oxyradicals with the polyunsaturated fatty acids of cellular phospholipids such as PC, and (c) reversible as long as the cells membrane repair functions (PC hydrolysis and biosynthesis) are sufficient to repair injured membranes. These results suggest that stretch‐induced cell injury in vitro may mimic in part experimental traumatic brain injury in vivo because alterations in cellular PC biosynthesis and PLC activity are similar in both models. Therefore, this in vitro model of stretch‐induced injury may supplement or be a reasonable alternative to some in vivo models of brain injury for determining the mechanisms by which traumatic cell injury results in cell dysfunction.

Hypolipidemic activity of in vitro inhibitors of hepatic and intestinal sn-glycerol-3-phosphate acyltransferase and phosphatidate phosphohydrolase

A number of agents including a series of 1,3-bis (substituted phenoxy)-2-propanones were screened in vitro for their ability to inhibit hepatic and intestinal microsomal sn-glycerol-3-phosphate acyltransferase and phosphatidate phosphohydrolase. Effective inhibitors reduced in vivo hepatic and intestinal glycerolipid production and with one exception also lowered serum triglyceride levels, suggesting that agents which inhibit potential rate-limiting steps of glycerolipid biosynthesis may be effective hypolipidemic agents. Two compounds, 1-methyl-4-piperidyl bis (p-chlorophenoxy) acetate (Sah 42-348) and 1,3-bis (p-methylphenoxy)-2-propanone were the best inhibitors of glycerolipid biosynthesis and lipid-lowering agents. The lipid-altering effects of both drugs were compared to chlorophenoxyisobutyrate during high fructose intake in rats. Each agent reduced fructose induced glycerolipid biosynthesis and serum triglyceride levels to similar degrees.

An enzymatic explanation of the differential effects of oleate and gemfibrozil on cultured hepatocyte triacylglycerol and phosphatidylcholine biosynthesis and secretion.

Incubation (1-4 h) of primary cultures of adult rat hepatocytes with gemfibrozil (0.1-1.0 mM) significantly decreased the: (1) incorporation of [1,3-14C]glycerol into cellular triacylglycerol (30%); (2) secretion of labeled (VLDL) triacylglycerol (4-fold); and (3) oleate-induced rise in triacylglycerol biosynthesis and secretion. Gemfibrozil also increased the: (1) incorporation of labeled glycerol into cellular phosphatidylcholine (2-fold); and (2) secretion of labeled (HDL) phosphatidylcholine (10-fold). The gemfibrozil-dependent increase in the flux of labeled diacylglycerol into phosphatidylcholine is rapid (15 min) and associated with a 2-fold increase in membrane-bound phosphocholine cytidylyltransferase activity. A phosphocholine cytidylyltransferase-mediated rise in cellular CDP choline content may explain the gemfibrozil-dependent rise in phosphatidylcholine biosynthesis since homogenates of monolayers incubated with CDP choline preferentially incorporate labeled diacylglycerol into phosphatidylcholine rather than triacylglycerol. Therefore, the triacylglycerol-lowering potential of gemfibrozil may be due in part to its ability to shunt liver cell diacylglycerol into phosphatidylcholine rather than triacylglycerol. These results suggest that CDP choline may be a key regulator of the diacylglycerol branchpoint, since diacylglycerol is primarily incorporated into phosphatidylcholine or triacylglycerol depending on whether CDP choline is or is not available.

The role of phospholipid metabolism in bromobenzene- and carbon tetrachloride-dependent hepatocyte injury

Sprague-Dawley rats and cultured rat hepatocytes exposed to bromobenzene (BB) and carbon tetrachloride (CCl4) display rapid and significant increases and decreases in hepatic phospholipase C (PLC) and sn-glycerol-3-phosphate acyltransferase (GPAT) activities, respectively. Primary cultures of adult rat hepatocytes were used to determine if the BB- and CCl4-dependent alterations in phospholipid metabolism were related to the hepatotoxicity of these agents. Cultured hepatocytes exposed to BB and CCl4 exhibited a rapid (1 to 5 min). PLC-mediated reduction (20 to 80%) in [32P]phosphatidylserine content. Other phospholipids were also reduced; however, phosphatidylserine was preferentially degraded by hepatotoxin-activated PLC. A time course of CCl4-and BB-induced cellular events showed that these agents (1) rapidly activate liver cell PLC activity; (2) accelerate 86Rb release; (3) decrease GPAT acyltransferase activity; and (4) cause a release of intracellular enzymes (GOT and GPT). All of these BB- and CCl4-mediated effects on the functional integrity of liver cells were blocked or reduced by agents (EDTA and chlorpromazine) that reduce the BB- and CCl4-dependent rise in PLC activity. Therefore, BB- and CCl4-dependent alterations in the functional and structural integrity of liver cells may be a result of accelerated phospholipid degradation and a corresponding inability of the cell to repair injured membranes by generating new phospholipids.

Studies of the formation and relase of glycerolipids by primary monolayer cultures of adult rat hepatocytes

Abstract Primary monolayer culture of adult rat hepatocytes represents a relatively novel and potentially useful in vitro technique for studying various hepatic processes for extended periods of time. This study evaluates the potential of hepatocyte monolayers for studying hepatic glycerolipid biosynthesis. Hepatocyte monolayers synthesize glycerolipids from [1,3- 14 C]glycerol and fatty acids and release them into the culture medium. Glycerolipid synthesis and release by monolayers declines 40 to 50% during the first 24 h of culture and these lower rates are maintained for at least 72 h. Triacylglycerol formation and release is dependent upon fatty acid concentration in the incubation medium although phospholipid is not. The release of triacylglycerol is greater in the presence of unsaturated acids (16 : 1, 18 : 1 and 18 : 2) than saturated acids (16 : 0 and 18 : 0) although saturated acids produce equal or greater amounts of intracellular triacylglycerol. Phospholipid and triacylglycerol release is reduced in monolayers exposed to colchicine for 24 h but only triacylglycerol release is lowered by the presence of cycloheximide. These results suggest release of triacylglycerol-containing lipoprotein by hepatocyte monolayers. However, we were unable to detect known apolipoprotein in the culture medium by immunologie techniques. Exposure of hepatocyte monolayers to ethanol and estradiol causes alterations in glycerolipid production and release. These changes in glycerolipid formation correlate well with corresponding alterations in glycerol kinase activity measured in monolayer homogenates. These experiments demonstrate the potential usefulness of hepatocytes in monolayer to study various aspects of glycerolipid metabolism. Their longer viability, days rather than hours, is an advantage over other in vitro liver preparations (slices, homogenates, isolated hepatocytes and liver perfusion) for many types of experiments.

The effects of bromobenzene and Carbon tetrachloride exposure in vitro on the phospholipase C activity of rat liver cells

Homogenates were prepared from isolated rat hepatocytes, previously incubated (30 to 120 min) in the presence of bromobenzene (BB), Carbon tetrachloride (CCl4), or dimethyl sulfoxide (DMSO = control vehicle). Homogenates of hepatocytes incubated with BB and CCl4 compared to those exposed to DMSO exhibited a rapid increase (two- to five-fold) in their capacity to convert labeled, membrane-bound phosphatidic acid into diacylglycerol. The BB- and CCl4-dependent increase in diacylglycerol (DG) content, at the expense of phosphatidate, was Ca2+ dependent and was attributed to changes in cell surface (plasma membrane) enzyme activity since the response occurred primarily in 1000g cell fractions. when either purified plasma membranes or 1000g cell fractions, isolated from fresh liver homogenates, were incubated with BB and CCl4 in vitro, a rise (two- to fourfold) in the conversion of labeled phosphatidate into DG was also observed. These Ca2+-dependent, BB- and CCl4-induced changes in hepatocellular glycerolipid content were caused by a BB- and CCl4-activated phospholipase C which degrades membrane phospholipids. This hypothesis is supported by the observation that 32P-labeled phospholipids of hepatocyte monolayers were rapidly catabolized (10 to 50%) after BB and CCl4 exposure (5 min). Also, there was an increased conversion of [methyl-14C]cytidine diphosphocholine into phosphatidylcholine after cell surface fractions were incubated with phospholipase C, BB, or CCl4, a change which reflects a rise in hepatocellular DG content. A rapid, BB- and CCl4-induced rise in hepatocellular phospholipase C activity that degrades cellular phospholipids could disrupt the structure and function of membranes and may represent an early event in toxic substance-related hepatocyte injury.

Effect of hydrazine exposure on hepatic triacylglycerol biosynthesis.

Acute hydrazine exposure elevated rat liver triacylglycerol content and produced a rapid rise in triacylglycerol production from sn-[1,3-14C]glycerol 3-phosphate by liver homogenate and microsomal fractions. Hydrazine treatment also increased the incorporation of [1,3-14C]glycerol into hepatic triacylglycerol by the intact animal. Homogenates of hepatocyte monolayers exposed to hydrazine in vitro also exhibited an increased capacity to form triacylglycerol from sn-[1,3-14C]glycerol 3-phosphate. Hydrazine-dependent increases in hepatic triacylglycerol production measured in vitro correlated well with an increase in microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) activity. Therefore, the fatty liver associated with hydrazine exposure may be explained in part by a rise in the enzymatic capacity of hepatic triacylglycerol biosynthesis.

The acute effects of streptozotocin-induced diabetes on rat liver glycerolipid biosynthesis.

Streptozotocin-induced diabetes produced a significant rise in rat serum and liver triacylglycerol content and hepatic triacylglycerol biosynthesis measured in vivo. Microsomes, isolated from the livers of streptozotocin-exposed animals (2-72h), exhibited an increased capacity to incorporate sn-[1,3-(14)C]glycerol 3-phosphate into neutral lipid (diacylglycerol and triacylglycerol) in the presence of ATP, CoA and palmitate. The streptozotocin-induced elevation of microsomal neutral lipid production was accompanied by a corresponding rise in the activity of microsomal phosphatidate phosphohydrolase (4-fold after 72 h of streptozotocin exposure). Diabetic-dependent increases in acylglycerol formation, phosphatidate phosphohydrolase activity and serum triacylglycerol and fatty acid levels were reversed by administering insulin (10 units protamine zinc/kg) at 16-h intervals (three separate doses( beginning 24 h after streptozotocin exposure. However, the diabetic-related rise in hepatic triacylglycerol content was only partially corrected by insulin administration. Streptozotocin-relate increases in liver triacylglycerol biosynthesis and phosphatidate phosphohydrolase activity we associated with alterations in plasma factors, since homogenates of hepatocyte monolayers exposed (18h) to plasma isolated from diabetic (72 h exposure to streptozotocin) animals exhibit an increased capacity to incorporate sn-[1,3-(14)C]glycerol 3-phosphate into triacylglycerol compared to homogenates of cells exposed to plasma from control (non-fasted) animals. The importance of these plasma factors in altering hepatic acylglycerol formation was also supported by the observation that hepatocyte monolayers exposed to a mixture of plasma isolated from normal (non-fasted) animals and plasma components elevated in diabetes (glucagon, glucose, oleate and ketones) showed increases in triacylglycerol formation which were similar to those produced by exposure to diabetic plasma. Additional studies demonstrated that fatty acids (oleate) appeared to be the agent primarily responsible for the diabetic plasma-induced rise in monolayer triacylglycerol biosynthesis and phosphatidate phosphohydrolase activity.

Toxic interactions between carbon tetrachloride and chloroform in cultured rat hepatocytes.

Primary cultures of adult rat hepatocytes were incubated (1.5-16 hr) with various concentrations of CCl4 (less than or equal to 0.5 mM) and/or CHCl3 (less than or equal to 2.5 mM). Agent-dependent alterations in hepatocyte functions were assessed by measuring (1) [3H]choline incorporation into phosphatidylcholine (endoplasmic reticulum), (2) MTT (tetrazolium salt) reduction (mitochondria), and (3) AST release into medium (plasma membrane). Cultured hepatocytes incubated with 0.5 mM CCl4 displayed a significant (p less than or equal to 0.001) and rapid (1.5 hr) reduction (40%) in endoplasmic reticulum function that preceded significant (p less than or equal to 0.001) alterations in mitochondria (6-16 hr) and plasma membrane (6-16 hr) functions. CCl4-dependent alterations in liver cell functions are a result of CCl4 bioactivation since metyrapone inhibits the CCl4-mediated changes in cell functions. Response surface methods (RSM) were used to determine the influence of combinations of CCl4 and CHCl3 on liver cell MTT reduction and [3H]choline incorporation. Regression coefficients were determined for CCl4, CHCl3, and CCl4-CHCl3. All results were significant (p less than 0.0001) and implied that CCl4 was a more potent hepatotoxin in vitro than CHCl3. The RSM analysis also suggested that combinations of CHCl3 and CCl4 have greater than additive effects on MTT reduction and [3H]choline incorporation. These effects of CCl4 and/or CHCl3 on liver cell functions in vitro are consistent with liver alterations observed in vivo. Therefore, primary cultures of adult rat hepatocytes may be an appropriate model in vitro to assess the hepatotoxic potential of agents alone or in combination.

The role of CCl4 biotransformation in the activation of hepatocyte phospholipase C in vivo and in vitro.

Rats treated with a single 0.5 ml/kg dose (ip) of CCl4 exhibited a threefold increase in liver microsomal phospholipase C (PLC) activity that was enhanced by phenobarbital and diminished by metyrapone pretreatment, respectively. Hepatocytes and hepatocellular fractions exposed to 0.5 mM CCl4 in vitro also exhibited a rapid rise in PLC activity that was reduced by metyrapone. Metyrapone also reduced the CCl4-related increase in the PLC-mediated reductions in cellular phosphatidylcholine content. The influence of CCl4 biotransformation on the activation of liver cell PLC was assessed in vitro. Covalent binding of 14CCl4 metabolites to isolated hepatocyte proteins and lipids was linear through 20 min of incubation and then quickly plateaued. The association of CCl4 metabolites with cellular phospholipids was inhibited by metyrapone and preceded the CCl4-dependent rise in PLC activity. CCl4-mediated increases in PLC activity were rapid and preceded reductions in cell viability. The translocation of cytosolic PLC to membranes such as the endoplasmic reticulum may explain the rapid, metabolite-dependent activation of PLC.PLC activation by haloalkanes was proportional to dose and incubation time in the order of CBrCl3 greater than CCl4 greater than CHCl3 greater than CFCl3 which corresponds to the observed hepatotoxic potential of these agents in vivo and in vitro. Haloalkane-dependent increases in PLC activity were inhibited by metyrapone. These results suggest that chemical metabolites activate PLC in vitro and in vivo. Therefore, the activation of a PLC that degrades membrane phospholipids may represent an important step in the pathogenic scheme of chemical-mediated liver cell necrosis.