scholarly journals Cyclic AMP accumulation in HeLa cells induced by cholera toxin. Involvement of the ceramide moiety of GM1 ganglioside

1990 ◽  
Vol 271 (1) ◽  
pp. 107-111 ◽  
Author(s):  
M Masserini ◽  
P Palestini ◽  
M Pitto ◽  
V Chigorno ◽  
M Tomasi ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Cholera Toxin ◽  
Molecular Species ◽  
Hela Cells ◽  
Gm1 Ganglioside ◽  
Cyclic Amp Accumulation ◽  
Long Chain Base ◽  
Ganglioside Species ◽  
Long Chain ◽  
Ceramide Moiety

The influence of ceramide composition on the rate of GM1 association to HeLa cells has been investigated by incubating the cells in the presence of either native ganglioside or molecular species carrying highly homogeneous long chain base moieties, fractionated from native GM1. The GM1 ganglioside species carrying the unsaturated C18 long chain base moiety proved to have the fastest rate of association, whereas the saturated species carrying 20 carbon atoms had the slowest rate. After having increased the GM1 cell content (65-fold) by incubation with the various ganglioside species, the cells were incubated with cholera toxin and the time course of cyclic AMP accumulation was monitored. Remarkable differences among cells enriched with the various molecular species were found in the duration of the lag time preceding the accumulation of cyclic AMP, the shortest being displayed by the unsaturated C18 species. Moreover, the amount of cyclic AMP accumulated after a given time of incubation with cholera toxin was significantly higher when the C18:1-GM1 species was present than with native GM1. Fluorescence anisotropy experiments, carried out using the probe 1,3-diphenylhexatriene, show that the GM1 ganglioside ceramide moiety was also modifying the cell membrane fluidity of the host.

scholarly journals Preparation of GM1 ganglioside molecular species having homogeneous fatty acid and long chain base moieties.

1985 ◽  
Vol 26 (2) ◽  
pp. 248-257
Author(s):  
S Sonnino ◽  
G Kirschner ◽  
R Ghidoni ◽  
D Acquotti ◽  
G Tettamanti
Keyword(s):  
Fatty Acid ◽  
Molecular Species ◽  
Gm1 Ganglioside ◽  
Chain Base ◽  
Long Chain Base ◽  
Long Chain

scholarly journals Bradykinin-dependent activation of adenylate cyclase activity and cyclic AMP accumulation in tracheal smooth muscle occurs via protein kinase C-dependent and -independent pathways

1994 ◽  
Vol 297 (1) ◽  
pp. 233-239 ◽  
Author(s):  
P A Stevens ◽  
S Pyne ◽  
M Grady ◽  
N J Pyne
Keyword(s):  
Smooth Muscle ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Phospholipase D ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Tracheal Smooth Muscle ◽  
Constitutive Activation ◽  
Cyclic Amp Accumulation

Treatment of cultured tracheal smooth-muscle cells (TSM) with phorbol 12-myristate 13-acetate (PMA) (100 nM) or bradykinin (100 nM) elicited enhanced basal and guanosine 5′-[beta gamma-imido]-triphosphate-stimulated adenylate cyclase activities in subsequently isolated membranes. Combined stimulation of cells was non-additive, indicating that both agents activate adenylate cyclase via similar routes. Both PMA (100 nM) and bradykinin (100 nM) allowed the alpha subunit of Gs to act as a more favourable substrate for its cholera-toxin-catalysed ADP-ribosylation in vitro. PMA was without effect on intracellular cyclic AMP in control cells. However, constitutive activation of Gs by treatment in vivo with cholera toxin (0.5 ng/ml, 18 h) sensitized the cells to PMA stimulation, resulting in a concentration-dependent increase in intracellular cyclic AMP accumulation (EC50 = 7.3 +/- 2.5 nM, n = 5). Bradykinin also elicited a concentration-dependent increase in intracellular cyclic AMP (EC50 = 63.3 +/- 14.5 nM, n = 3). Constitutive activation of Gs resulted in an increased maximal response (10-fold) and potency (EC50 = 6.17 +/- 1.6 nM, n = 3) to bradykinin. This response was not affected by the B2-receptor antagonist, NPC567 [which selectively blocks bradykinin-stimulated phospholipase C (PLC), with minor activity against phospholipase D (PLD) activity]. Des-Arg9-bradykinin (a B1-receptor agonist) was without activity. These results suggest that the receptor sub-type capable of activating PLD may also be stimulatory for cyclic AMP accumulation. Furthermore, pre-treatment of the cells with butan-l-ol (0.3%, v/v), which traps phosphatidate derived from PLD reactions, blocked the bradykinin-stimulated increase in intracellular cyclic AMP. These studies suggest that there may be a causal link between PLD-derived phosphatidate and the positive modulation of adenylate cyclase activity. In support of this, the concentration-dependence for bradykinin-stimulated adenylate cyclase activity was identical with that of bradykinin-stimulated phospholipase D activity (EC50 = 5 nM). Bradykinin, but not PMA, was also capable of eliciting the inhibition of cyclic AMP phosphodiesterase activity in TSM cells (EC50 > 100 nM) via an unidentified mechanism. These studies indicate that cross-regulation between the cyclic AMP pathway and phospholipid-derived second messengers in TSM cells does not occur as a consequence of PLC-catalysed PtdIns(4,5)P2 hydrolysis, but may involve, in part, PLD-catalysed phosphatidylcholine hydrolysis.

scholarly journals Incorporation and metabolism of exogenous GM1 ganglioside in rat liver

1986 ◽  
Vol 237 (1) ◽  
pp. 147-155 ◽  
Author(s):  
R Ghidoni ◽  
M Trinchera ◽  
B Venerando ◽  
A Fiorilli ◽  
S Sonnino ◽  
...  
Keyword(s):  
Rat Liver ◽  
Degradation Process ◽  
Major Product ◽  
The Other ◽  
Gm1 Ganglioside ◽  
Lysosomal Fraction ◽  
Long Chain Base ◽  
Terminal Galactose ◽  
Subcellular Level ◽  
Long Chain

The pathways of metabolic processing of exogenously administered GM1 ganglioside in rat liver was investigated at the subcellular level. The GM1 used was 3H-labelled at the level of long-chain base ([Sph(sphingosine)-3H]GM1) or of terminal galactose ([Gal-3H]GM1). The following radioactive compounds, derived from exogenous GM1, were isolated and chemically characterized: gangliosides GM2, GM3, GD1a and GD1b (nomenclature of Svennerholm [(1964) J. Lipid Res. 5, 145-155] and IUPAC-IUB Recommendations [(1977) Lipids 12, 455-468]); lactosylceramide, glucosylceramide and ceramide; sphingomyelin. GM2, GM3, lactosylceramide, glucosylceramide and ceramide, relatively more abundant shortly after GM1 administration, were mainly present in the lysosomal fraction and reflected the occurrence of a degradation process. 3H2O was also produced in relevant amounts, indicating complete degradation of GM1, although no free long-chain bases could be detected. GD1a and GD1b, relatively more abundant later on after administration, were preponderant in the Golgi-apparatus fraction and originated from a biosynthetic process. More GD1a was produced starting from [Sph-3H]GM1 than from [Gal-3H]GM1, and radioactive GD1b was present only after [Sph-3H]GM1 injection. This indicates the use of two biosynthetic routes, one starting from a by-product of GM1 degradation, the other implicating direct sialylation of GM1. Both routes were used to produce GD1a, but only the first one for producing GD1b. Sphingomyelin was the major product of GM1 processing, especially at the longer times after injection, and arose from a by-product of GM1 degradation, most likely ceramide.

scholarly journals Human fibroblasts in culture metabolize differently exogenous G(M3) ganglioside species containing C18 and C20 sphingosine.

1998 ◽  
Vol 45 (2) ◽  
pp. 385-392
Author(s):  
V Chigorno ◽  
M Valsecchi ◽  
S Sonnino
Keyword(s):  
Human Fibroblasts ◽  
Decisive Role ◽  
Metabolic Fate ◽  
Base Content ◽  
Long Chain Base ◽  
Complex Lipids ◽  
Sphingolipid Biosynthesis ◽  
Ganglioside Species ◽  
Long Chain ◽  
Complex Sphingolipids

Preparation of radioactive GM3 species containing isotopically labeled C18 sphingosine or C20 sphingosine is reported and their use for studying some aspects of the sphingolipid biosynthesis in cells is discussed. Human fibroblasts in culture that have only C18 sphingolipids and GM3 as the major gangliosides, were fed with the two radioactive GM3 species. The radioactive gangliosides were taken up by the cells and metabolized. The analyses of the radioactivity metabolic fate, in this model provides the following information. i--About 70-80% of the total catabolic sphingosine is re-cycled for biosynthesis of complex sphingolipids. ii--A small amount of the catabolic C20 sphingosine was re-cycled for biosynthesis of C20 sphingolipids, thus yielding complex lipids that are not naturally present in fibroblast cells. iii--A regulatory step in the biosynthesis of sphingolipid species differring long chain base content, C18 or C20 sphingosine, is in some way involved in the first steps of sphingolipid biosynthesis, and thus plays a decisive role in the availability of the long chain bases.

scholarly journals Different metabolic recycling of the lipid components of exogenous sulphatide in human fibroblasts

1988 ◽  
Vol 252 (2) ◽  
pp. 375-379 ◽  
Author(s):  
M Trinchera ◽  
U Wiesmann ◽  
M Pitto ◽  
D Acquotti ◽  
R Ghidoni
Keyword(s):  
Fatty Acid ◽  
Human Fibroblasts ◽  
Total Lipid Extract ◽  
Phospholipid Biosynthesis ◽  
Metabolic Fate ◽  
Long Chain Base ◽  
Cultured Human Fibroblasts ◽  
Almost All ◽  
Long Chain ◽  
Ceramide Moiety

Cultured human fibroblasts were fed with two differently labelled sulphatide molecules [one labelled on C-3 of the sphingosine (Sph) moiety [(Sph-3H]sulphatide), the second on C-1 of stearic acid [(stearoyl-14C]sulphatide)], and the intracellular metabolic fate of radioactivity was monitored. Incorporated radioactivity was almost all recovered in the total lipid extract, regardless of the labelling position of the added sulphatide; however, large differences in the level of incorporation occurred among labelled glycosphingolipids. For example, sphingomyelin was present as the major radiolabelled lipid after [Sph-3H]-sulphatide incubation, but was detectable only in trace amounts after [stearoyl-14C]sulphatide administration; in the latter case the radioactivity was located predominantly in glycerophospholipids. From this finding it can be inferred that the free long-chain base (sphingosine) that originates from lysosomal catabolism of sulphatide is mainly, and quite specifically, utilized for sphingomyelin biosynthesis, whereas the ceramide moiety is not; conversely the fatty acid released from ceramide is non-specifically re-utilized for phospholipid biosynthesis.

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