Biomedical subjects

C S Hii

Publications and source records attributed to C S Hii.

At least 37 records · Page 2Linked to original sources

PubMed1996

Effect of tumor necrosis factor-alpha on the metabolism of arachidonic acid in human neutrophils.

Although tumor necrosis factor-alpha (TNF-alpha) has been shown to induce marked changes in the physiology/pathophysiology of cells, little is known about the effects of this cytokine on cellular lipid metabolism. In this study we examined the effects of TNF-alpha on the metabolism of eicosatetraenoic acid (arachidonic acid, (20:4(n-6)) in human neutrophils. Pretreatment of neutrophils with TNF-alpha caused a rapid increase in the incorporation of [1-14C]20:4(n-6) substrate into cellular phosphatidylinositol and phosphatidic acid and a slower rise in the incorporation into phosphatidylcholine and phosphatidylethanolamine. Radioactivity was exclusively associated with the sn-2 position of each molecule. The labeling pattern of other phospholipids, neutral lipids, and eicosanoids was unchanged. TNF-alpha had no effect on the distribution of radioactivity in 1-acyl, 1-alkyl, and 1-alk-1-enyl subclasses of phosphatidylcholine, phosphatidylethanolamine, and triglyceride. Chain elongation, beta-oxidation and desaturation of [1-14C]20:4(n-6) were not modulated by the cytokine. TNF-alpha stimulated the release of [3H]20:4(n-6) from prelabeled neutrophils and also induced the production of endogenous unesterified 20:4(n-6). Concomitantly, treatment with the cytokine caused a decrease in the mass of cellular phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine and an increase in the levels of corresponding lysophospholipids, but had no significant effect on sphingomyelin, phosphatidic acid, diglyceride, and other lipids. TNF-alpha did not evoke neutrophils prelabeled with [3H]lyso platelet activating factor to produce [3H]phosphatidylethanol, [3H]phosphatidic acid, or [3H]diglyceride in the presence of ethanol, indicating that phospholipases D and C were not activated. Treatment of the leukocytes with the cytokine had no effect on the activity of neutral and acidic sphingomyelinase. These data collectively provide evidence that TNF-alpha specifically induces the turnover of neutrophil phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine, which are enriched with 20:4(n-6) by the activation of phospholipase A2.

Arachidonic Acid↗

PubMed1995

Polyenoic very-long-chain fatty acids mobilize intracellular calcium from a thapsigargin-insensitive pool in human neutrophils. The relationship between Ca2+ mobilization and superoxide production induced by long- and very-long-chain fatty acids.

Fatty acids with more than 22 carbon atoms (very-long-chain fatty acids; VLCFAs) are normal cellular components that have been implicated in the pathophysiology of a number of peroxisomal disorders. To date, however, essentially nothing is known regarding their biological activities. Ca2+ mobilization is an important intracellular signalling system for a variety of agonists and cell types. Given that several polyunsaturated long-chain fatty acids mobilize intracellular Ca2+ and that we have postulated that the VLCFAs may be involved in signal transduction, we examined whether the tetraenoic VLCFA induced Ca2+ mobilization in human neutrophils. We report that fatty acid-induced intracellular Ca2+ mobilization declined for fatty acid species of more than 20 carbon atoms, but increased again as the carbon chain length approached 30. This Ca2+ mobilization occurred independently of inositol 1,4,5-triphosphate production and protein kinase C translocation and involved both the release of Ca2+ from the intracellular stores and changes to the influx or efflux of the ion. We further observed that triacontatetraenoic acid [30:4(n-6)] mobilized Ca2+ from a thapsigargin-insensitive intracellular pool distinct from the thapsigargin-sensitive pools affected by arachidonic acid [20:4(n - 6)] or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). 20:4 (n - 6) induced strong superoxide production (chemiluminescence) which was inhibited by thapsigargin pretreatment. In contrast, fatty acid-induced superoxide production progressively declined as the carbon chain length increased beyond 20-22 carbon atoms. Further studies suggested that the thapsigargin-insensitive Ca2+ mobilization elicited by 30:4 (n - 6) was not related to oxyradical formation, while the thapsigargin-sensitive Ca2+ mobilization induced by 20:4 (n - 6) may be involved in the initiation but not necessarily the maintenance of superoxide production. In conclusion, this is the first report to demonstrate a biological activity for the VLCFA and indicates that 30:4 (n - 6) influences second messenger systems in intact cells that differ from those affected by long-chain fatty acids such as 20:4 (n - 6).

Calcium↗

PubMed1995

Activation of mitogen-activated protein kinase by arachidonic acid in rat liver epithelial WB cells by a protein kinase C-dependent mechanism.

Arachidonic acid (20:4(n-6)), which is released by cells responding to a wide range of stimuli, may play an important role in intracellular signaling. We now report that incubation of WB cells with 20:4(n-6) resulted in the appearance of several tyrosine-phosphorylated cytosolic proteins. Two of the phosphotyrosine-containing proteins, migrating in SDS-polyacrylamide gels of approximately 43 and 45 kDa, corresponded in mobility to phosphorylated species of the 42- and 44-kDa mitogen-activated protein kinase (MAPK) isoforms. Immunoblots of soluble fractions from unstimulated WB cells with anti-MAPK antibodies revealed the presence of the 42- and 44-kDa isoforms of MAPK. Upon incubation with 20:4(n-6), the mobility of both isoforms was retarded, consistent with their activation by phosphorylation. Chromatography of soluble fractions from these cells on Mono Q columns revealed early and late eluting peaks of myelin basic protein kinase activity, which contained the 42- and 44-kDa MAPK isoforms, respectively. Activation of MAPK was transient, peaking at 5 min, and was detectable at 5 microM 20:4(n-6). Further studies into the mechanisms by which MAPK was activated by 20:4(n-6) strongly suggested the involvement of protein kinase C (PKC). Not only did incubation of WB cells with 20:4(n-6) result in the translocation of PKC alpha, delta, and epsilon to a particulate fraction, it was found that the fatty acid failed to activate MAPK in cells pretreated for 26 h with phorbol 12-myristate 13-acetate, which depleted WB cells of PKC alpha, delta and epsilon. In addition, fatty acids of the n-3 series were effective activators of MAPK. The present study, to our knowledge, is the first to report that polyunsaturated fatty acids can cause the activation of MAPK.

Animals↗

PubMed1995

Inhibition of gap junctional communication by polyunsaturated fatty acids in WB cells: evidence that connexin 43 is not hyperphosphorylated.

Polyunsaturated fatty acids have attracted much interest due to their wide spectrum of biological activities which include the modulation of gap junctional communication (GJC). Since gap junctions play critical roles in maintaining the functional integrity of organs and tissues, and loss of intercellular communication is associated with a number of pathological conditions, we investigated the effects of the n-6 and n-3 series of polyunsaturated fatty acids and their derivatives on GJC in WB cells as determined by the ability of Lucifer Yellow-loaded cells to transfer the dye to neighbouring recipient cells. Studies were also conducted to investigate the possible mechanisms of action of the fatty acids. Treatment of cells with 10 microM arachidonic acid (20:4 n-6) resulted in a rapid and transient loss of communication competence. The response to 20 microM 20:4 (n-6) was prolonged (> 210 min) but was readily reversible by washing the cells with fatty acid-free bovine serum albumin. Cells which had regained their communication competence responded to further additions of 20:4 (n-6). The fatty acids, 18:3 (n-6), 20:5 (n-3), 22:6 (n-3) and the 15-hydroxy- and the 15-hydroperoxy-derivatives of 20:4 (n-6) were also powerful inhibitors of GJC, while 23:4 (n-6) was a relatively weak inhibitor. The saturated 20 carbon fatty acid, 20:0, and the methyl ester of 20:4 (n-6) were without effect. This illustrates the importance of unsaturation and the carboxyl group as structural requirements for activity. 20:4 (n-6)-induced inhibition of dye transfer was not attenuated by pretreating the cells with either phorbol-12-myristate-13-acetate (PMA) or indomethacin, suggesting that regulation of gap junctional permeability by 20:4 (n-6) in WB cells was neither dependent on PMA-responsive isozymes of protein kinase C nor required the metabolism of the fatty acids by cyclo-oxygenase. However, the effect of 20:4 (n-6) was antagonized by preincubating WB cells with either nordihydroguaiaretic acid or (+/-)-isoproterenol and isobutylmethyl-xanthine. Western blot analysis of connexin 43 (Cx43), the major gap junctional protein expressed in these cells, revealed no detectable changes to the electrophoretic mobility of Cx43 even after 60 min of incubation in the presence of 20:4 (n-6). As expected, other inhibitors of gap junctional permeability including epidermal growth factor, phorbol ester or lysophosphatidic acid induced a retardation in the mobility of Cx43, indicating an enhancement in the phosphorylation of Cx43 protein.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed1994

Neutrophil migration inhibitory properties of polyunsaturated fatty acids. The role of fatty acid structure, metabolism, and possible second messenger systems.

The n-3 polyunsaturated fatty acids (PUFA) appear to have antiinflammatory properties that can be partly explained by their biological activity on leukocytes. Since leukocyte emigration is an essential component of the inflammatory response, we have examined the effects of the n-3 PUFA (eicosapentaenoic and docosahexaenoic acids) on neutrophil random and chemotactic movement. Preexposure of neutrophils for 15-30 min to 1-10 micrograms/ml PUFA reduced the random and chemotactic migration to both FMLP- and fungi-activated complement. The inhibitory effect diminished with increasing saturation and carbon chain length, and methylation abolished this activity. Arachidonic and docosahexaenoic acids were the most active fatty acids. The PUFA concentration required to inhibit migration was dependent on cell number, suggesting that the fatty acid effects on leukocyte migration in vivo may be governed by the stage of the inflammatory response. It was concluded that the PUFA rather than their metabolites were responsible for the inhibition since: (a) antioxidants did not prevent the PUFA-induced migration inhibition and the hydroxylated intermediates were less active, and (b) inhibitors of the cyclooxygenase and lipoxygenase pathways were without effect. Inhibitors of protein kinases and calmodulin-dependent enzyme system did not prevent the PUFA-induced migration inhibition, which was also independent of phospholipase D-catalyzed hydrolysis of phospholipids. It is also shown that PUFA decrease the FMLP-induced Ca2+ mobilization.

Calcium↗

PubMed1992

Basal and phorbol-ester-stimulated turnover of phosphatidylcholine in HeLa cells involve different pathways.

HeLa cells prelabelled with [3H]lysoplatelet-activating factor (lyso-PAF) accumulated phosphatidic acid (PtdOH) when incubated in the presence of either propranolol (an inhibitor of PtdOH phosphohydrolase) or phorbol ester. In the presence of ethanol, phorbol ester but not propranolol stimulated the accumulation of phosphatidylethanol, an index of phospholipase D activity. Incubation of cells with [3H]lyso-PAF led to a rapid accumulation of label in diacylglycerol (DG) followed by a delayed accumulation in PtdOH. It is concluded that propranolol-induced PtdOH accumulation is derived from DG by DG kinase and does not involve phospholipase D.

Cell Membrane↗

PubMed1992

Tumour promoting phorbol esters activate phospholipase D in mouse skin.

Phospholipase D catalyses a transphosphatidylation reaction in the presence of primary alcohols, resulting in the formation of phosphatidylalcohol derivatives. In the present work we show that application of phorbol esters and butanol to mouse skin causes the rapid accumulation of phosphatidylbutanol (PBol), indicating the activation of phospholipase D. A similar accumulation of PBol was observed when the skin was treated with phorbol esters in vivo and skin pieces incubated with butanol in vitro. PBol formation was stimulated by the active tumour promoters 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein and phorbol-12,13-didecanoate (PDD) but not by the inactive promoter 4 alpha-PDD. Accumulation of PBol was not observed 24 h after application of TPA, a treatment which has been shown to deplete epidermal protein kinase C activity.

Animals↗

PubMed1991

Phorbol ester-stimulated hydrolysis of phosphatidylcholine and phosphatidylethanolamine by phospholipase D in HeLa cells. Evidence that the basal turnover of phosphoglycerides does not involve phospholipase D.

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulated the release of [3H]ethanolamine from HeLa cells prelabeled with [3H]ethanolamine within 2 min, and of [3H]choline from cells prelabeled with [3H]choline after a lag of 10-20 min. This result suggests that TPA activates phospholipase D. Propranolol alone or propranolol plus TPA stimulated phosphatidic acid (PA) labeling in cells prelabeled with [3H]hexadecanol. In the presence of ethanol, TPA stimulated the accumulation of labeled phosphatidylethanol (PEth); no PEth was formed in the absence of TPA. TPA-dependent PEth accumulation was not observed in cells pretreated with TPA to down-regulate protein kinase C, whereas propranolol-induced accumulation of PA was unaffected by TPA pretreatment. Incubation of prelabeled cells with propranolol alone caused a rapid loss of label and phospholipid mass from both phosphatidylethanolamine and phosphatidylcholine (PC) together with an accumulation of PA and phosphatidylinositol plus phosphatidylserine. When [3H]hexadecanol-prelabeled cells were pulse labeled with 32P to label nucleotide pools, propranolol induced the accumulation of both 3H- and 32P-labeled PA. When cells were prelabeled with lyso-PC double labeled with 3H and 32P, and incubated with propranolol, only 3H-labeled PA accumulated, indicating that the pathways involved in the basal turnover of PC resulted in the loss of 32P from the lipid. These results suggest that the basal turnover of phosphatidylethanolamine and PC involves the sequential actions of phospholipase C, diglyceride kinase, and PA phosphohydrolase.

Choline↗

PubMed1990

Phorbol esters modulate the turnover of both ether- and ester-linked phospholipids in cultured mammalian cells.

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the metabolism of ester- and ether derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were studied in HeLa and HEL-37 cells. TPA stimulated the incorporation of [3H]choline into diacyl-, alkylacyl- and alkenylacy/PC in HeLa cells, but inhibited the incorporation of [3H]ethanolamine into the corresponding derivatives of PE. TPA also stimulated the incorporation of [3H]ethanolamine into lysoPE and the release of labelled ethanolamine and phosphoethanolamine from HeLa cells prelabelled with [3H]ethanolamine. All responses to TPA were abolished in HeLa cells preincubated with the phorbol ester and which were deficient in protein kinase C. In HEL-37 cells TPA stimulated label incorporation into both ester- and ether-forms of PE. The marked effects of TPA on ether-lipid metabolism raises the possibility that hydrolysis products of this class of lipid are important in transmembrane signalling pathways.

Animals↗

PubMed1989

Phorbol esters stimulate a phospholipase D-catalysed reaction with both ester- and ether-linked phospholipids in HeLa cells.

The phorbol ester, 12-tetradecanoylphorbol-13-acetate (TPA), stimulated the accumulation of labelled phosphatidylethanol (PEth) in HeLa cells prelabelled with [3H]hexadecanol and incubated in the presence of ethanol. PEth formation involves a phospholipase D-catalysed phosphatidyl transferase reaction. [3H]Hexadecanol labelled both ester- and ether-derivatives of HeLa cell phospholipids and TPA stimulated the conversion of both classes of lipid to PEth.

Ethers↗

PubMed1988

DNA-binding proteins in protein kinase C preparations.

Human DNA enriched in repetitive sequences specifically bound to a component(s) in purified preparations of rat brain protein kinase C (PKC). DNA which bound to protein was cloned in pUC-19 and one clone characterized as containing an approx. 140 bp insert. The band containing this insert (separated by acrylamide gel electrophoresis) was lost when the DNA was incubated with purified PKC preparations. Thus a protein in relatively pure PKC preparations is a sequence-selective DNA-binding protein. The results raise the possibility that PKC or a fragment of PKC binds selectively to specific DNA sequences.

Animals↗

PubMed1988

Role of protein kinase C in arginine-induced glucagon secretion from isolated rat islets of Langerhans.

This study investigated the effect of pretreatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA) on arginine-induced glucagon secretion. Isolated islets of Langerhans were pretreated by culturing for 18-24 h in the presence of 200 nM of the tumour-promoting phorbol ester PMA or 200 nM of the non-tumour-promoting phorbol ester 4-phorbol didecanoate (PDD). Islets pretreated with PMA did not secrete glucagon in response to 0.1 or 1 microM PMA on subsequent incubation, in contrast to PDD-pretreated islets which responded significantly on subsequent incubation with PMA. Pretreatment with PMA led to impairment of arginine-induced glucagon secretion. PMA-pretreated islets permeabilized by high-voltage discharge retained their normal secretory responses to calcium and cyclic AMP, but had an impaired secretory response to PMA. These results suggest (1) that protein kinase C (PKC) is likely to be present in the A cell, (2) that short-term culture in tumour-promoting phorbol ester leads to down-regulation of PKC, (3) that the PKC pathway is involved in arginine-induced glucagon secretion and (4) that pretreatment does not effect the A cell response to other intracellular mediators.

Animals↗

PubMed1987

A re-assessment of the role of protein kinase C in glucose-stimulated insulin secretion.

Isolated rat islets of Langerhans which had been pretreated with 200 nM-phorbol 12-myristate 13-acetate (PMA) for 20-24 h, a treatment reported in other cell types to deplete cells of protein kinase C activity, were found not to contain detectable Ca2+/phospholipid-dependent protein kinase activity. These islets did not secrete insulin in response to a subsequent exposure to PMA (0.1 or 1 microM) during a 30 min incubation, although insulin secretion could be stimulated by 20 mM-glucose, a response which was enhanced by 20 microM-forskolin. PMA-pretreated islets that had been permeabilized by high-voltage discharge showed unimpaired secretory responses to an increase in Ca2+ concentration, cyclic AMP and forskolin. These results suggest that (i) pretreatment of islets with tumour-promoting phorbol esters may be a useful means of investigating the role of protein kinase C in stimulus-secretion coupling in the pancreatic beta-cell and (ii) protein kinase C may not play an essential role in glucose-induced insulin secretion.

Animals↗

PubMed1987

Role of second messengers in the regulation of glucagon secretion from isolated rat islets of Langerhans.

The roles of diacylglycerol (DAG), cAMP and Ca2+ in mediating the stimulatory action of arginine on pancreatic A cells have been investigated using phorbol esters, forskolin, a Ca2+ ionophore and trifluoroperazine (TFP). 0.5 microM 4 beta-phorbol 12-myristate 13-acetate (PMA) which stimulated glucagon secretion by approximately 3-fold in the absence of arginine, was unable to enhance arginine-stimulated glucagon secretion. Higher concentrations (1 and 10 microM) of PMA were able to enhance glucagon secretion in the presence of 1.25, 2.5, 5 but not 10 mM arginine. Insulin secretion was enhanced by PMA under all the conditions tested. Arginine (10 and 20 mM)-stimulated secretion of glucagon and insulin were synergistically augmented by 20 microM forskolin. While the effects of forskolin plus PMA on the A cells were additive, the effects of the two agents on the B cells were synergistic. The responses of the A and B cells to arginine required extracellular Ca2+. Secretion of the two hormones was dose-dependently stimulated by A23187. Arginine-stimulated glucagon secretion but not insulin secretion, was dose-dependently inhibited by TFP. These results suggest that proposed cellular second messengers interact differently in the A and B cells, and DAG and Ca2+ may play pivotal roles in mediating the actions of arginine on the A but not B cells; cAMP may play a modulatory role in the A cell response to arginine.

Animals↗

PubMed1986

Enhancement of glucagon secretion from isolated rat islets of Langerhans by phorbol 12-myristate 13-acetate.

The phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA), at concentrations of 0.1 microM and above, stimulated secretion of glucagon and of insulin from isolated rat islets of Langerhans incubated in the presence of 5.5 mM-glucose. Stimulation of secretion of both hormones by 1 microM-PMA persisted in the absence of external Ca2+, and could be abolished by incubating the islets at 4 degrees C. These findings suggest a role of protein kinase C in the alpha-cell (and beta-cell) secretory mechanism.

Animals↗

PubMed1986

Restoration of the A cell response to glucose in isolated rat islets of Langerhans.

This study investigated the modulatory effects of forskolin, phorbol 12-myristate 13-acetate (PMA) and arginine on pancreatic glucagon secretion in response to changes in glucose concentrations. Glucose, on its own (0, 5, 9 and 18 mM), did not modify glucagon secretion from A cell-rich isolated rat islets of Langerhans. In the presence of 20 microM forskolin, glucagon release was stimulated dose-dependently on lowering the external glucose concentration to 0 mM. Sensitivity to glucose was achieved in the presence of either PMA or arginine; both agents also significantly enhanced glucagon release at all glucose concentrations tested. The response of the B cells in these experiments were as expected from the available literature. These results indicate that the endogenous rate of glucagon secretion in the isolated islet preparation was minimal and was insensitive to glucose, sensitivity of the A cells to glucose could be restored by either arginine or agents which alter the concentration or activity of proposed cellular second messengers.

Animals↗

PubMed1985

Assessment of the antidiabetic activity of epicatechin in streptozotocin-diabetic and spontaneously diabetic BB/E rats.

(-)-Epicatechin has previously been suggested to rapidly reverse alloxan diabetes in rats. We have assessed the therapeutic value of the compound in two further animal models of insulin-dependent diabetes mellitus, namely streptozotocin-diabetic rats and the spontaneously diabetic BB/E rat. There was no indication of a reversal of established diabetes in either the streptozotocin-diabetic or the spontaneously diabetic BB/E rats. Moreover, epicatechin also failed to halt the progression of the disease in prediabetic BB/E rats. Earlier claims of the potential use of epicatechin as an antidiabetic agent must therefore be treated with some caution.

Administration, Oral↗

PubMed1985

Effects of flavonoids on insulin secretion and 45Ca2+ handling in rat islets of Langerhans.

The effects of some flavonoids, a group of naturally occurring pigments one of which has been claimed to possess antidiabetic activities, on insulin release and 45Ca2+ handling have been studied in isolated rat islets of Langerhans. Insulin release was enhanced by approximately 44-70% when islets were exposed to either (-)epicatechin (0.8 mmol/l) or quercetin (0.01-0.1 mmol/l); others such as naringenin (0.1 mmol/l) and chrysin (0.08 mmol/l) inhibited hormone release by approximately 40-60%. These effects were observed only in the presence of 20 mmol glucose/l. Quercetin (0.01 mmol/l) and (-)epicatechin (0.8 mmol/l) both inhibited 45Ca2+ efflux in the presence and absence of extracellular Ca2+. In the presence of 20 mmol glucose/l both the short-term (5 min) and steady-state (30 min) uptake of 45Ca2+ were significantly increased by either quercetin or (-)epicatechin. These results suggest that the stimulatory compounds such as quercetin and (-)epicatechin may, at least in part, exert their effects on insulin release via changes in Ca2+ metabolism.

Animals↗