Jolanta Barańska

Influence of temperature on the composition of fatty acids and on lipogenesis in frog tissues

1. 1. Frogs (Rana esculenta) were adapted to various temperatures and the fatty acid composition of tissues and lipogenesis from radioactive precursors were studied. The activities of NADPH-generating enzyme systems were also compared. 2. 2. Liver and adipose tissue from cold-adapted frogs contained more unsaturated fatty acids and less saturated, than the corresponding tissues from frogs kept at higher temperatures. However, differences were found mostly in the proportions of polyenoic acids and not in those of the monoenoic acids. The changes in fatty acid composition of phospholipids and of neutral lipids were similar. 3. 3. As a result of in vitro lipogenesis from 1-C14-acetate mostly saturated and monoenoic fatty acids were formed and there was no indication of de novo formation of linoleic acid in tissues from either cold-adapted frogs or those kept at 20°C. 4. 4. The temperature of adaptation did not influence either the extent of hepatic lipogenesis from C14-acetate or the distribution of radioactivity in lipid fractions. On the other hand, in adipose tissue of frogs kept at 20°C both lipogenesis and esterification of newly formed fatty acids were strongly inhibited as compared with the tissue from cold-adapted animals. 5. 5. No correlation between the rate of lipogenesis and the activities of NADPH-generating enzymes could be observed and under the conditions of depressed lipogenesis formation of NADPH did not seem to be rate limiting.

Two subtypes of G protein‐coupled nucleotide receptors, P2Y1 and P2Y2 are involved in calcium signalling in glioma C6 cells

In glioma C6 cells, the stimulation of P2Y receptors by ADP, ATP and UTP initiated an increase in the intracellular Ca2+ concentration, in a process that involved the release of Ca2+ from InsP3‐sensitive store and the capacitative, extracellular Ca2+ entry. The presence of external Ca2+ was not necessary to elevate Ca2+. The rank order of potencies of nucleotide analogues in stimulating [Ca2+]i was: 2MeSADP > ADP > 2MeSATP = 2ClATP > ATP > UTP. α,β‐Methylene ATP, adenosine and AMP were ineffective. ADP and UTP effects were additive, while actions of ATP and UTP were not additive on [Ca2+]i increase. Similarly, cross‐desensitization between ATP and UTP but not between ADP and UTP occurred. Suramin, a non‐specific nucleotide receptors inhibitor, antagonized ATP‐, UTP‐ and ADP‐evoked Ca2+ responses. PPADS, a selective antagonist of the P2Y1 receptor‐generated InsP3 accumulation, decreased ADP‐initiated Ca2+ response with no effect on ATP and UTP. Pertussis toxin (PTX) reduced ADP‐ and ATP‐induced Ca2+ increases. Short‐term treatment with TPA, inhibited both ATP and ADP stimulatory effects on [Ca2+]i. ADP inhibited isoproterenol‐induced cyclic AMP accumulation. PTX blocked this effect, but PPADS did not. RT – PCR analysis revealed the molecular identity of P2Y receptors expressed by glioma C6 cells to be both P2Y1 and P2Y2. It is concluded that both P2Y1 and P2Y2 receptors co‐exist in glioma C6 cells. ADP acts as agonist of the first, and ATP and UTP of the second one. Both receptors are linked to phospholipase C (PLC).

ADP-evoked phospholipase C stimulation and adenylyl cyclase inhibition in glioma C6 cells occur through two distinct nucleotide receptors, P2Y1 and P2Y12

In this study we characterized the subtypes of nucleotide P2Y receptors that respond to ADP in glioma C6 cells. Direct visualization of phosphatidylinositol 4,5‐bisphosphate at the cell surface revealed that extracellular ADP activates phospholipase C (PLC). Knock‐down of P2Y1 receptor with antisense oligonucleotide, as well as treatment with MRS2179 and pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (P2Y1 antagonists), attenuates receptor‐mediated PLC activity. Adenylyl cyclase inhibition by ADP remains unchanged under these conditions. Reverse transcription‐PCR analysis showed that P2Y12 receptor is expressed in C6 cells. We therefore conclude that, in glioma C6 cells, two P2Y receptor subtypes are present: P2Y1, coupled to PLC, and P2Y12, negatively coupled to adenylyl cyclase.

Differential effects of P2Y1 and P2Y12 nucleotide receptors on ERK1/ERK2 and phosphatidylinositol 3-kinase signalling and cell proliferation in serum-deprived and nonstarved glioma C6 cells

We have previously shown that, in glioma C6 cells, two nucleotide ADP‐sensitive receptors coexist: P2Y1, coupled to PLC and responsible for Ca2+ release, and P2Y12, negatively coupled to adenylate cyclase. In the present study, we examined the effects of the stimulation of these two receptors on ERK1/2 and PI3‐K activation, and cell proliferation in either serum‐deprived or nonstarved C6 cells. In response to ADP and its analogues, in serum‐starved cells, both p44 ERK1 and p42 ERK2 were activated in a time‐dependent manner, as monitored by Western blot analysis using an antiphospho‐p42/p44 MAPK antibody. The phosphorylation was reduced both by removal of the extracellular Ca2+ and partially or almost completely by MRS2179 or AR‐C69931MX, specific antagonists of the P2Y1 and P2Y12 receptors, respectively. The inhibitory effect of antagonists was additive. These data indicate the involvement of both receptors, P2Y1 and P2Y12, in the ERK1/2 activation, but the P2Y12 receptor contribution predominates. ERK1/2 activity was positively correlated with cell proliferation of cultured glioma C6 cells. In nonstarved cells, ADP markedly decreased the PI3‐K activity. In contrast, in serum‐starved cells, ADP evoked an increase in the PI3‐K activity. Blocking of the P2Y1 receptor by MRS2179 additionally increased this ADP response. These results suggest that the P2Y1 receptor has an inhibitory and the P2Y12 receptor a stimulatory effect on PI3‐K signalling pathway. RT–PCR analysis revealed different mRNA expression of both receptors in starved and nonstarved cells. In nonstarved cells, the P2Y1 receptor mRNA predominates, whereas in serum‐deprived cells the expression of P2Y12 mRNA becomes more pronounced.

Exchange of phospholipids between microsomes and mitochondrial outer and inner membranes

Abstract 1. 1. 32P-labelled phospholipids are transferred from rat liver microsomes to rat liver mitochondria when the particles are incubated in the presence of the soluble cytoplasmic fraction. Labelled phospholipids are incorporated mainly into the outer mitochondrial membrane, and only after disruption or detachment of the outer membrane could a substantial uptake of the label by the inner membrane be observed. The exchange of phospholipids between microsomes and the outer mitochondrial membrane was essentially complete within 15–20 min under experimental conditions, whereas the exchange between microsomes and exposed inner mitochondrial membranes proceeded for at least 60 min. 2. 2. Free fatty acids added to the mitochondrial suspension in tracer amounts could easily penetrate across the outer membrane and be bound by the inner membrane. 3. 3. Liver slices incubated in the presence of [14C]choline incorporated the label into both microsomes and mitochondria. A chase applied after 60 min labelling produced a decrease of the specific activity of microsomal lecithin, but the specific activity of mitochondrial lecithin continued to increase. [14C]Choline was mainly incorporated into lecithin of the outer mitochondrial membrane, about as little as 20 % being found in the inner membrane. 4. 4. The results are interpreted in the sense that the exchange of phospholipids between the inner and outer membranes of intact mitochondria either does not occur or is qualitatively different and quantitatively much slower than a direct exchange between microsomes and the outer mitochondrial membrane.

Inhibition of phosphatidylserine synthesis by glutamate, acetylcholine, thapsigargin and ionophore A23187 in glioma C6 cells

Phosphatidylserine synthesis was studied in glioma C6 cells with [14C]serine and in the presence or absence of agents which increase the level of [Ca2+]i. It was found that glutamate and acetylcholine inhibited this synthesis by up to 40%, whereas thapsigargin and the ionophore A23187 inhibited by up to 70%. The inhibitory effect of thapsigargin and the A23187 was observed in Ca(2+)-free medium. The data show that the inhibition of this synthesis is caused by the Ca(2+)-depletion from endoplasmic reticulum, suggesting that the synthesis of phosphatidylserine occurs on the luminal side of these structures and can be regulated by transmembrane signaling systems.

Role of the actin cytoskeleton in store-mediated calcium entry in glioma C6 cells

The effects of actin cytoskeleton disruption by cytochalasin D and latrunculin A on Ca2+ signals evoked by ADP, UTP or thapsigargin were investigated in glioma C6 cells. Despite the profound alterations of the actin cytoskeleton architecture and cell morphology, ADP and UTP still produced cytosolic calcium elevation in this cell line. However, calcium mobilization from internal stores and Ca2+ influx through store-operated Ca2+ channels induced by ADP and UTP were strongly reduced. Cytochalasin D and latrunculin A also diminished extracellular Ca2+ influx in unstimulated glioma C6 cells previously incubated in Ca2+ free buffer. In contrast, the disruption of the actin cytoskeleton had no effect on thapsigargin-induced Ca2+ influx in this cell line. Both agonist- and thapsigargin-generated Ca2+ entry was significantly decreased by the blocker of store-operated Ca2+ channels, 2-aminoethoxydiphenylborate. The data reveal that two agonists and thapsigargin activate store-operated Ca2+ channels but the mechanism of activation seems to be different. While the agonists evoke a store-mediated Ca2+ entry that is dependent on the actin cytoskeleton, thapsigargin apparently activates an additional mechanism, which is independent of the disruption of the cytoskeleton.

Adaptor Protein Ruk/CIN85 is Associated with a Subset of COPI-Coated Membranes of the Golgi Complex

Regulator of ubiquitous kinase/Cbl‐interacting protein of 85 kDa (Ruk/CIN85) and CD2‐associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS) comprise a family of vertebrate adaptor proteins involved in several important cellular processes, including downregulation of activated receptor tyrosine kinases, regulation of cytoskeletal rearrangements, phosphatidylinositol 3‐kinase (PI 3‐kinase) signalling and apoptosis. The role of Ruk/CIN85 as a scaffold protein involved in membrane trafficking processes has been demonstrated in model cell systems. However, intracellular localization of endogenous Ruk/CIN85 has never been comprehensively assessed. We carried out detailed studies of subcellular distribution of Ruk/CIN85 in adherent cultured human cells using antibodies that recognize distinct epitopes of the protein and revealed a punctate immunostaining pattern, common for proteins involved in intracellular trafficking processes. Our data indicate that Ruk/CIN85 is distributed between several different membrane trafficking compartments, but the major pool of Ruk/CIN85 is associated with the Golgi complex, mainly with a subpopulation of COPI‐coated vesicles involved in retrograde endoplasmic reticulum‐Golgi and intra‐Golgi transport. This localization pattern is dependent on the integrity of Golgi complex and intact microtubular network. Only a small pool of Ruk/CIN85 is present in compartments involved in clathrin‐mediated endocytosis and sorting. These results suggest that endogenous Ruk/CIN85 may be involved in regulation of specific membrane trafficking processes.

Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells.

We characterized the expression and functional properties of the ADP‐sensitive P2Y1 and P2Y12 nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long‐term serum starvation, cell morphology changed from fibroblast‐like flat to round, the adhesion pattern changed, cell‐cycle arrest was induced, extracellular signal‐regulated kinase (ERK1/2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y12 receptor relative to P2Y1 was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y1 receptor was low, and the P2Y12 receptor was the major player, responsible for ADP‐evoked signal transduction. The P2Y12 receptor activated ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR‐C69931MX, a specific antagonist of the P2Y12 receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y1 receptor, indicating the inhibitory role of P2Y1 in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y1 to P2Y12 would appear to be a new and important self‐regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation.

Esterification of fatty acids in the wax moth haemolymph and its possible role in lipid transport

Abstract Haemolymph of wax moth larvae (Galleria mellonella L.) was incubated with 1-14C-palmitate at 37°C for various periods. Gradual esterification of free fatty acids occurred in the haemolymph concomitantly with hydrolysis of some ester bonds. Incorporation of 14C-palmitate took place predominantly in the triglyceride fraction of the haemolymph lipids. Very little radioactivity was found in the diglycerides, which represent the largest fraction of the glycerides in the haemolymph of the larvae. Incorporation of 14C-palmitate was strongly inhibited by sodium chloride and sodium fluoride, known inhibitors of lipoprotein lipase and pancreatic (‘true’) lipase, respectively. The incorporation of labelled fatty acid was found to take place chiefly at the α-position of the glyceride molecule. The process of esterification may represent an exchange reaction between free fatty acids and glyceride fatty acids, catalysed by lipases present in the haemolymph.