Growth-dependent accumulation of monoalkylglycerol in Madin-Darby canine kidney cells. Evidence for a role in the regulation of protein kinase C.

1-O-Alkyl-sn-glycerol (alkylglycerol) forms the backbone of complex ether-linked glycerolipids, including biologically active lipids such as platelet-activating factor. Synthetic alkylglycerol itself possesses several potent pharmacological activities and has been shown to inhibit protein kinase C (PKC) in vitro. In spite of these properties, free alkylglycerol has been regarded only as a potential product of the inflammatory degradation of complex ether lipids rather than a natural cell constituent. To explore the possibility that endogenous alkylglycerol functions as a physiological regulator in normal cells, we measured its content, along with related monoglycerides and diglycerides, by high performance liquid chromatography and gas-liquid chromatography in Madin-Darby canine kidney (MDCK) cells. The content of free alkylglycerol increased up to 20-fold during the growth of MDCK cell cultures to a confluent density. The increase was greatest during the log phase of growth, in which the content of alkylglycerol rose from 6.0 +/- 1.3 nmol/10(8) cells in preconfluent cultures to 23.6 +/- 3.4 nmol/10(8) cells in confluent cultures. Analysis of the molecular species of alkylglycerol showed that the higher content in quiescent MDCK cells was due primarily to an increase in 1-O-octadecyl-sn-glycerol. In contrast, the levels of monoacylglycerol and the PKC activator diacylglycerol were lower in confluent, quiescent cultures than in preconfluent, proliferating cultures. A similar pattern of changes in the monoglyceride and diglyceride content was observed in interleukin-3-dependent CFTL-12 mast cells when cell proliferation was blocked by growth factor withdrawal. Growth of MDCK cells to a confluent density resulted in a decrease in particulate PKC enzyme activity to a level that was only 6% of that in proliferating cells. To explore whether the accumulation of cellular alkylglycerol contributes to growth-dependent changes in PKC activity, we examined the effects of adding alkylglycerol to the activity and subcellular distribution of the enzyme in MDCK cells. Treatment of cells with 1-O-dodecyl-sn-glycerol resulted in a decrease in the activity of membrane-associated PKC activity and inhibited 12-O-tetradecanoylphorbol-13-acetate-stimulated translocation of PKC from the cytosol to the membrane fraction. Alkylglycerol was also shown to inhibit the activity of purified PKC in vitro when present at levels similar to that of the diacylglycerol activator. We propose that the accumulation of alkylglycerol during the growth of MDCK cells to a confluent density contributes to the decrease in PKC activity. The control of cellular alkylglycerol levels may be a novel mechanism for the regulation of cellular physiology.

Inhibition of phorbol ester-stimulated arachidonic acid release by alkylglycerols.

Although synthetic analogs of alkylglycerol (AG), such as dodecylglycerol, possess potent biological activities, their mechanism of action has not been determined. We recently detected substantial amounts of AG in unstimulated MDCK cells (Warne, T.R. and Robinson, M. (1991) Anal. Biochem. 198, 302-307) raising the possibility that the endogenous compound may act as a biological mediator. In this study, we examined the effects of synthetic AG on the release of arachidonic acid and arachidonate metabolites (AA) from Madin Darby canine kidney (MDCK) cells in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) in order to characterize its effects on this signalling pathway. Treatment of MDCK with AG potently inhibited the release of AA during subsequent stimulation with TPA. Dodecylglycerol, the most effective of a series of alkyglycerols tested, was active at concentrations as low as 3 microM. The sn-1 and sn-3 forms of AG were found to be equally potent inhibitors. The effects of AG on AA release were not the result of arachidonic acid redistribution among cellular lipids and were independent of the phospholipid source of the released AA. AG did not inhibit the release of AA from MDCK cells when bradykinin was used as a stimulus, indicating selectivity for the effects produced by phorbol esters. These results show that AG can function as a potent and specific inhibitor of TPA-mediated AA release. The ability of AG to regulate this signalling pathway in intact MDCK cells, together with its natural occurrence, suggests a potential bioregulatory role for the endogenous compound as an inhibitor of protein kinase C.

Accumulation of fatty alcohol in MCF-7 breast cancer cells.

The MCF-7 cell line (human breast epithelial cells) accumulates fatty alcohols. The fatty alcohols were identified as C16:0, C18:0, and C18:1 alcohols by thin-layer chromatography and gas chromatography/mass spectrometry. This accumulation of alcohols in MCF-7 was found in cultures of MCF-7 cells obtained from other laboratories but not in a variety of unrelated cell lines. The presence of the alcohols suggested an aberrant ether lipid metabolism in the MCF-7 cells. Therefore, the capacity for either lipid biosynthesis was evaluated using cells incubated with either [14C]stearyl alcohol or [14C]stearic acid. MCF-7 cells incorporated less than 0.4% of the [14C]alcohol into ether-linked phospholipids, whereas the AB589 breast epithelial cells, used as a "normal control" for comparisons, did not accumulate fatty alcohol and incorporated approximately 20% of the radiolabeled alcohol into phospholipids containing ether linkages. Although the MCF-7 cells were unable to effectively incorporate the fatty alcohol into ether linkages, the cells were able to oxidize the alcohol to fatty acid. When incubated with [14C]stearic acid, the conversion to radiolabeled fatty alcohol in MCF-7 cells was approximately four times higher than the alcohol levels found in AB589 cells. While deficient in the ability to synthesize ether linkages, the MCF-7 cells did incorporate radiolabeled hexadecylglycerol, a precursor containing an ether linkage, into phospholipids. Collectively, the data indicate that the MCF-7 cells possess a deficiency in the alkyl DHAP synthase activity. A near absence of ether-linked lipids in the MCF-7 cells was indicated by the radiolabeling studies, and this finding was corroborated by results from HPLC analysis. Analyses of the partial glycerides, obtained from the enzymatic hydrolysis of cellular phospholipids, found only trace levels of ether lipids in the MCF-7 cells. The aberration in ether lipid biosynthesis did not correlate with the expression of the multidrug resistance phenotype in a series multidrug resistant MCF-7 variants. The results are discussed relative to the use of the MCF-7 cells as a model for investigations of ether lipid biosynthesis and the cellular physiology of ether lipids.

A method for the simultaneous determination of alkylacylglycerol, diacylglycerol, monoalkylglycerol, monoacylglycerol, and cholesterol by high-performance liquid chromatography.

We describe a method for the quantitative analysis of the individual subclasses (1-O-alkyl and 1-acyl) of diradylglycerols and monoradylglycerols. These lipids, along with cholesterol, were separated from other neutral and polar lipids on silica columns and analyzed by normal-phase high-performance liquid chromatography (HPLC) as their benzoate derivatives. Cholesterylbenzoate, alkylacylglycerolbenzoate, diacylglycerolbenzoate, monoalkylglyceroldibenzoate, and monoacylglyceroldibenzoate eluted from HPLC in five distinct zones. The derivatives of diradylglycerols and monoradylglycerols were further separated within each discrete zone on the basis of the total number of aliphatic carbons at the sn-1 and sn-2 positions. Radiolabeled cholesterol and dihexadecanoylglycerol were used to monitor recovery. Amounts of synthetic alkylacylglycerol, diacylglycerol, monoalkylglycerol, and monoacylglycerol as low as 0.2 nmol per subclass could be accurately quantified. The technique was used to determine the content of diradylglycerol and monoradylglycerol subclasses in Madin-Darby canine kidney and CFTL-12 mast cells. This method should prove useful for the quantitation of lipid second messengers in cultured cells.

IL-3-induced generation of alkylacylglycerol and diacylglycerol in an IL-3-dependent cell line.

The hematopoietic growth factor IL-3 promotes the proliferation and development of several hematopoietic lineages. Inasmuch as protein kinase C has been suggested to mediate the response of IL-3, we examined the accumulation of diradylglycerols (DG) in response to IL-3 in CFTL-12 cells, a murine mast cell line that requires IL-3 for growth. Exposure of CFTL-12 cells to IL-3 resulted in the conversion of [3H]myristate-labeled lipids to DG. Mass analysis of the DG of CFTL-12 cells cultured in the presence of IL-3 showed that 58% was the ether-linked form, alkylacylglycerol, and 42% was diacylglycerol. The levels of both alkylacylglycerol and diacylglycerol declined when CFTL-12 cells were withdrawn from IL-3 and became quiescent. Stimulation of quiescent cells with IL-3 produced an acute increase in the mass of both alkylacylglycerol and diacylglycerol, consistent with phosphatidylcholine as a significant source. The effects of PMA on the generation of DG were examined to explore the role of protein kinase C activation in the response to IL-3. PMA stimulated an increase in DG accumulation that was not augmented by the simultaneous addition of IL-3. Down-modulation of protein kinase C by long term PMA treatment reduced, but did not eliminate, the IL-3-stimulated increase in DG, suggesting that protein kinase C activation results in an amplification of the initial accumulation of DG. These results indicate a role for DG, generated through the hydrolysis of phosphatidylcholine, in the induction of protein kinase C activity and the events leading to cell proliferation in response to IL-3.

Sources of diradylglycerols generated during cell growth and phorbol ester stimulation in Madin-Darby canine kidney cells.

The molecular species of diacylglycerol and alkylacylglycerol of Madin-Darby canine Kidney (MDCK) cells were analyzed to determine the sources of diradylglycerols generated during cell growth and phorbol ester stimulation. MDCK cells in log phase growth contained higher levels of diacylglycerol and alkylacylglycerol than confluent cells. Both subclasses of diradylglycerol showed higher levels of saturated and monoenoic species during log phase. Glycerol incorporation into diradylglycerols was increased during growth, consistent with an increase in their synthesis de novo. Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, caused an increase in the level of diacylglycerol but not alkylacylglycerol. Log phase MDCK cells showed a greater response to TPA treatment than confluent cells. The molecular species of diacylglycerol generated during stimulation with either TPA or dioctanoylglycerol closely resembled the species of phosphatidylcholine. These results indicate that TPA and synthetic diacylglycerol stimulate endogenous diacylglycerol production through the hydrolysis of phosphatidylcholine. In contrast, the higher content of diacylglycerol and alkylacylglycerol in replicating MDCK cells is the result of an increase in their synthesis de novo.

Selection of a mutation conferring high NaCl tolerance to gametophytes of Ceratopteris.

Spores from a weakly salt tolerant strain of Ceratopteris richardii containing the mutation stl1 were irradiated and sown on nutrient medium supplemented with 200 mM NaCl. A single highly salt tolerant gametophyte was recovered and selfed to generate a homozygous sporophyte. Spores from this strain, 10α23, were used to document the sexual transmission of the trait and to monitor the inheritance of tolerance in crosses to both the wild type and to the parental salt tolerant strain. Genetic analysis showed the 10α23 strain to possess both the original stl1 mutation and an additional semi-dominant nuclear mutation, stl2, that individually conferred a high level of tolerance to gametophytes. In combination, both mutations had additive effects. Tolerance was also evident in sporophytes, but at a lower level than in gametophytes.

A method for the quantitative analysis of molecular species of alkylacylglycerol and diacylglycerol.

We describe a method for the quantitative analysis of molecular species of diacylglycerol and alkylacylglycerol as their diradylglycerobenzoate derivatives. Synthetic internal standards were used to provide quantitative determinations of the low levels of diacylglycerol and alkylacylglycerol and their individual molecular species in cultured cells. Diradylglycerols were isolated by thin-layer chromatography (TLC), converted to their benzoate derivatives and separated into subclasses by TLC. The molecular species of each subclass were analyzed by reversed-phase high performance liquid chromatography. Thirty-six species of diglyceride-type molecules were identified in Madin-Darby canine kidney cells. These cells were shown to contain 7.88 nmoles of diacylglycerol and 3.97 nmoles of alkylacylglycerol per mumole of phospholipid. Both subclasses contain predominantly monoenoic and saturated species. This technique should be valuable for studies examining the origin and metabolism of these important intracellular mediators.

Evidence for a gibberellin biosynthetic origin of ceratopteris antheridiogen.

The species-specific chemical messenger, antheridiogen A(Ce), mediates the differentiation of male gametophytes in the fern Ceratopteris. In order to investigate the biochemical origin of antheridiogen, the effect of the inhibitors, 2'-isopropyl-4'-(trimethylammoniumchloride)-5' -methylphenylpiperidine-1-carboxylate (AMO-1618), 2-chloroethyl trimethylammonium chloride (CCC), and alpha-cyclopropyl-alpha-(4-methoxyphenyl)-5-pyrimidine methyl alcohol (ancymidol) on gametophytic sex expression was determined in C. richardii. Both AMO-1618 and ancymidol blocked the production of male gametophytes in three genetically defined strains of C. richardii that exhibit different sensitivities to antheridiogen. Antheridiogen supplementation overcame inhibition by AMO-1618 and ancymidol, except in one strain (HaC18) that is insensitive to antheridiogen supplementation. These data suggest that the synthesis of Ceratopteris antheridiogen, a taxon that is insensitive to exogenously supplied gibberellins, occurs via a pathway that may include steps in common with gibberellin biosynthesis or involves similar reactions.

(2-Chloroethyl)phosphonic Acid Promotes Germination of Immature Spores of Ceratopteris richardii Brongn.

Freshly collected spores of strain Hn-n of Ceratopteris richardii Brongn. require storage for several months before attaining maximum germination rate. Treatments using (2-chloroethyl)phosphonic acid increased germination rate in freshly collected spores and decreased germination rate in older spores.