Miriam H. Fukami

On the mechanism of induction of the enzyme systems for peroxisomal β-oxidation of fatty acids in rat liver by diets rich in partially hydrogenated fish oil

In this paper, we describe the early biochemical changes in liver cells that occur in rats fed a semisynthetic diet containing 20% (w/w) partially hydrogenated fish oil. Within hours the level of ornithine decarboxylase (ODC) increased, peaked at about 24 h (11-fold increase) and returned to subnormal levels within 48 h. The diet evoked a similar rapid increase in the cellular level of mRNA for the bifunctional enzyme of peroxisomal beta-oxidation (enoyl-CoA hydratase: beta-hydroxyacyl-CoA dehydrogenase (HD)) (12-fold), followed by increases in the specific content of HD protein (3-fold) and the capacity for beta-oxidation in peroxisomes (5.3-fold). The cellular level of long-chain acyl-CoA increased 2.1-fold. By contrast, no significant changes were observed in the specific activities of ornithine decarboxylase, peroxisomal beta-oxidation activity and microsomal omega-hydroxylation as well as the level of long-chain acyl-CoA in livers of rats fed (1 week) diets containing 20% (w/w) soybean oil with added 3 or 6% (w/w) of either elaidic acid (18:1(11) (trans)), brassidic acid (22:1(13) (trans)) or erucic acid (22:1(13) (cis)). Expression of normal levels of mRNA for the bifunctional enzyme was also found. Morphometric analyses revealed no proliferation of peroxisomes in these fatty acid-supplemented diets, in contrast to that observed with the partially hydrogenated fish oil diet. These results are consistent with the proposal (Flatmark, T., Christiansen, E.N. and Kryvi, H. (1983) Biochim. Biohys. Acta 753, 460-466) that components in dietary oils, different from C22:1 cis and trans fatty acids, are responsible for the pleiotropic responses evoked in target cells. Thus, the pattern of response induced by partially hydrogenated fish oil mimics those induced by xenobiotic compounds collectively termed peroxisome proliferators.

The stimulatory effects of cationic amphiphilic drugs on human platelets treated with thrombin

The actions of eight cationic amphiphilic drugs on human platelets displayed three different effects according to drug concentration ranges. At lower concentrations (below approximately 25 microM), the drugs stimulated secretory responses induced by 0.2 U/mL of thrombin, while at concentrations in the 25-50 microM range they inhibited these responses. Above 50-100 microM, the drugs caused permeabilization of the platelet plasma membrane as measured by leakage of cytoplasmic adenine nucleotides. The effects of these agents on phosphoinositide metabolism were monitored in platelets prelabeled with (32)P-inorganic phosphate, such that phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP(2)), but not phosphatidylinositol (PI), were labeled to equilibrium. In unstimulated platelets, the level of labeled PA decreased slightly (about 25%), with corresponding increases in PIP(2) labeling up to drug concentrations of about 50 microM. In contrast to the relatively small changes in PI and PIP(2), the levels of labeled PIP, precursor to PIP(2), increased 2- to 4-fold in both resting and thrombin-treated platelets from 5 microM up to about 50-100 microM of drugs and remained elevated throughout the permeabilization concentrations. [(32)P]PA increased 20-fold over control upon thrombin activation and 5-30 microM of drugs caused [(32)P]PA to increase 30-37 times over that seen in control, resting platelets; the concentration of drugs that potentiated thrombin-induced [(32)P]PA elevation corresponded to that causing the potentiation of platelet secretion. Higher drug concentrations decreased [(32)P]PA elevation. [(32)P]PIP(2) levels increased about 25% in response to thrombin treatment alone; low concentrations of drugs led to another 25% elevation. A significant decrease in [(32)P]PIP(2) was seen above 30 microM, corresponding to inhibition of platelet secretion. Concentrations of 5-30 microM of several psychoactive agents, both neuroleptics and antidepressants, potentiated the thrombin-induced activation of platelets as measured by dense granule secretion and increased turnover of phosphoinositides. Remarkably, all of the drugs increased the levels of PIP even in resting platelets, indicating that they have common effects apart from the specific receptor interactions currently attributed to them. These common effects, e.g. an increase in membrane fluidity such as is known to be caused by amphipathic agents, may be in part responsible for the observed overlapping psychotropic effects of tricyclic antidepressants and phenothiazines.

Studies on catalase compartmentation in digitonin-treated rat hepatocytes

The cellular compartmentation of catalase was studied in digitonin-permeabilized rat hepatocytes. A biphasic dose-response curve was observed for the unmasking of catalase activity by digitonin in latency studies. About 40-60% of the total catalase activity was seen in the range of 5-200 microM digitonin compared to 13% free activity in control preparations without digitonin. The free catalase activity began to increase again above 300 micron digitonin, and all latency was lost around 500 microM and above. These results indicate that there exists in rat hepatocytes catalase with two levels of crypticity to digitonin, only one of which was seen in a mixed organelle preparation containing peroxisomes.

Differential secretion of blood platelet storage granules

Platelets contain three types of secretory granules, dense granules, α-granules and lysosomes, which are characterized by their different contents. Dense granule and α-granule secretion appear to be similar in responsiveness to dose and types of agonists, whereas lysosomal secretion is observed only with higher doses of strong agonists such as thrombin. Recently, with the advent of flow cytometry, surface expression of membrane granule proteins, which are claimed to be specific for granule type, has come into use as a monitor for secretion. Expression of CD62 (PADGEM) in particular has become synonymous with α-granule secretion, based on comparisons with measurements of β-thromboglobulin release by a method in which secretion is not stopped by fixation. We have now developed an immunoassay for fibrinogen that tolerates fixation stopping and have compared the release of dense and α-granule markers in the same platelet supernatants with the expression of CD62 and CD63 in gel-filtered platelets. At thrombin concentrations less than 0.04 U/ml, secretion of α-granule fibrinogen was both more rapid and quantitatively greater than that of dense granule serotonin, ATP and ADP. Comparison of the secretion of granule markers (contents) with the expression of granule membrane markers on the platelet surface showed that surface expression of CD62 (P-selectin, PADGEM) corresponded to fibrinogen secretion, and CD63 correlated reasonably well with the release of dense granule contents. Pretreatment of platelets with acetylsalicylic acid (ASA) before gel-filtration moderately inhibited thrombin-induced dense and α-granule release in GFP at a concentration range of 0.01-0.03 U/ml. The agonist effect of a thrombin receptor agonist peptide (TRAP) was comparable to that of thrombin with respect to all measured markers except for β-hexosaminidase release, which was significantly less with TRAP.

Glycerophospholipid metabolism : back to the future

It has become customary to regard the various glycerophospholipids as quite similar, and the acyl groups are considered to have little influence on the behaviour of the lipids in membranes or metabolism. Nevertheless, a number of recent observations by the authors and others indicate a high degree of metabolic compartmentation and substrate specificity with regard to the acyl substituents (acyl specificity) of glycerophospholipid metabolising enzymes in intact cells. 1. [32P]Orthophosphate and [3H]glycerol are incorporated into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of platelets and Swiss 3T3 fibroblasts with a [32P]/[3H]-ratio several fold lower than in glycerol-3-phosphate, phosphatidic acid (PA) and phosphatidylinositol (PI), suggesting distinct metabolic separation (probably by cellular compartmentation) of the glycerol and choline (or ethanolamine) branches of de novo phospholipid biosynthesis. 2. In fibroblasts the [32P]/[3H]-ratio varied 50-fold among the molecular species of PC, PE, PI and PA, which indicates that the enzymes involved in these conversions have some degree of acyl specificity. 3. In vitro assays for lipid-converting enzymes employ detergents, which affect acyl specificity of the enzymes (lipid kinases) both by their chemical nature and concentrations. 4. Thrombin stimulation of platelets causes formation of a multitude of diacylglycerol (DAG) molecular species, but only one major molecular species of PA is formed indicating that the DAG kinase may have distinct acyl specificity in the intact cell. 5. However, this specificity could also result from the net reactions of DAG kinase(s) and PA phosphohydrolase(s), which would constitute an ATP-utilising, paired regulation of the molecular species of PA and the inositol lipids on one hand, and PC, PE phosphatidylserine and triacylglycerol on the other. These findings indicate a high complexity of glycerophospholipid metabolism and a distinct acyl specificity in intact cells that are not apparent from studies in vitro. A major challenge for future research in this area is to bridge the apparent discrepancy between in vivo and in vitro observations regarding glycerophospholipid metabolism, an endeavour that will require more knowledge about the physical chemistry of naturally occurring molecular species than is available today. The most prevailing appreciation of glycerophospholipids among biological scientists to-day is that they can be distinguished functionally, topographically and metabolically only by their head groups and that they form the bilayer in biological membranes. Most of us know that the fatty acid in the sn-2 position is unsaturated and have been indoctrinated that the higher the degree of unsaturation, the greater the fluidity of the membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of a peptide binding to receptor for activated C-kinase (RACK1) inhibits phorbol myristoyl acetate-stimulated phospholipase D activity in C3H/10T1/2 cells: dissociation of phospholipase D-mediated phosphatidylcholine breakdown from its synthesis.

The C3H/10T1/2 Cl8 HAbetaC2-1 cells used in this study express a peptide with a sequence shown to bind receptor for activated C-kinase (RACK1) and inhibit cPKC-mediated cell functions. Phorbol myristoyl acetate (PMA) strongly stimulated phosphatidylcholine (PtdCho)-specific phospholipase D (PLD) activity in the C3H/10T1/2 Cl8 parental cell line, but not in Cl8 HAbetaC2-1 cells, indicating that full PLD activity in PMA-treated Cl8 cells is dependent on a functional interaction of alpha/betaPKC with RACK1. In contrast, the PMA-stimulated uptake of choline and its subsequent incorporation into PtdCho, were not inhibited in Cl8 HAbetaC2-1 cells as compared to Cl8 cells, indicating a RACK1-independent but PKC-mediated process. Increased incorporation of labelled choline into PtdCho upon PMA treatment was not associated with changes of either CDP-choline: 1,2-diacylglycerol cholinephosphotransferase activity or the CTP:phosphocholine cytidylyltransferase distribution between cytosol and membrane fractions in Cl8 and Cl8 HAbetaC2-1 cells. The major effect of PMA on the PtdCho synthesis in C3H/10T1/2 fibroblasts was to increase the cellular uptake of choline. As a supporting experiment, we inhibited PMA-stimulated PtdH formation by PLD, and also putatively PtdH-derived DAG, in Cl8 cells with 1-butanol. Butanol did not influence the incorporation of [(14)C]choline into PtdCho. The present study shows: (1) PMA-stimulated PLD activity is dependent on a functional interaction between alpha/betaPKC and RACK1 in C3H/10T1/2 Cl8 fibroblasts; and (2) inhibition of PLD activity and PtdH formation did not reduce the cellular uptake and incorporation of labelled choline into PtdCho, indicating that these processes are not directly regulated by PtdCho-PLD activity in PMA-treated C3H/10T1/2 Cl8 fibroblasts.

Biochemical properties of platelet microparticle membranes formed during exocytosis resemble organelles more than plasma membrane

Studies of [3H]glycerol turnover in phosphatidylcholine (PC) in platelets revealed two metabolic pools, a ‘low turnover PC’ in collagen‐induced microparticles with specific radioactivity only 10% of that found in the ‘high turnover PC’ of bulk platelet PC. Isolated organelle fractions of [3H]glycerol‐labelled platelets contained [3H]PC with specific radioactivities about 20% of that in membrane fractions. These results together with studies on distribution of concanavalin A‐FITC and GPlb, a plasma membrane receptor, indicate that microparticles formed during exocytosis are not simple vesiculations of plasma membrane, but they seem rather to originate from a relatively metabolically static membrane pool not accessible to extracellular reagents.

EDTA inhibits collagen-induced ATP+ADP secretion and tyrosine phosphorylation in platelets independently of Mg 2+ chelation and decrease in pH

The effects of Mg 2+ and EDTA on collagen-induced platelet dense granule secretion and protein tyrosine phosphorylation were studied in the presence and absence of various inhibitors of autocrine agonists that are released from secretory granules. Addition of EDTA to gel-filtered platelets in Tyrodes solution caused a decrease in pH of 0.7-0.9 pH units, due to chelation of Mg 2+ , and a marked inhibition of cATP+ADP secretion induced by collagen (25 w g/ml). Lowering pH of the platelet suspension to the same extent by HCl had no effect on secretion which also was the same in the absence of exogenous Mg 2+ as in its presence. Similarly, secretion induced by collagen per se , i.e., isolated from autocrine agonists by inhibitors, which was about 50% less than with autocrine agonists, was not affected by the presence or absence of exogenous Mg 2+ . The level of tyrosine phosphorylation of several proteins in resting platelets was higher in the absence of Mg 2+ than in its presence, but the onset of collagen-induced phosphorylation and dephosphorylation was more rapid in the absence of Mg 2+ . Tyrosine phosphorylation of p38 was specifically enhanced in the presence of inhibitors of autocrine phosphorylation and even more enhanced in the absence of Mg 2+ . EDTA inhibited the protein phosphorylation to the same extent in the presence as in the absence of exogenous Mg 2+ . These results show that EDTA inhibits collagen-induced dense granule secretion neither through chelation of Mg 2+ nor lowering of pH, and thus probably through other effects on the platelet by the EDTA2- species that are reflected in protein tyrosine phosphorylation signalling.

Identification of minor metabolites of phospholipid signal molecules in [ 32 P]P i -labelled platelets

Incubation of blood platelets with 32 P-labelled inorganic phosphate for 60 min leads to incorporation of radioisotope mainly into phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP) and phosphatidyl-4,5-bisphosphate (PIP 2 ) in resting platelets and into phosphatidic acid (PA) in activated platelets. Small amounts of other important phosphoinositide isomers also become labelled following platelet activation, among them the 3-phosphorylated derivatives. In addition, several other faintly labelled spots are visible on TLC separations. Three of these lipids have now been identified as lysophosphatidylinositol (lysoPI), lysophosphatidic acid (lysoPA) and CDP-diacylglcerol (CDP-DAG).[ 32 P]LysoPI was present in resting and activated platelets, whereas [ 32 P]lysoPA and [ 32 P]CDP-DAG were observed only upon platelet activation. The phosphoinositide cycle turns over without accumulation of [ 32 P]PA and [ 32 P]CDP-DAG in resting platelets. A large increase (as much as 40-fold) in the steady-state level of [ 32 P]PA is seen in thrombin-activated platelets. A slight increase in the steady-state levels of [ 32 P]CDP-DAG is accompanied by a similar increase in [ 32 P]PI and larger increases in [ 32 P]PIP and [ 32 P]PIP 2 (about 50%), which is indicative of a general increase in flux in the PPI cycle. Elevation of CDP-DAG levels is probably only a reflection of increased flux, whereas lysoPA and lysoPI have been reported to have diverse signalling functions in various cells.