BIOCHEMICAL
Vol. 85, No. 4, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
December 29,1978
NEUTRAL GLYCOSPHINGOLIPIDS
1314-1317
OF THREE CELL TYPES ISOLATED FROM GUINEA PIG GASTRIC MUCOSA
Jean-Franfois INSERM U 45 Pavillon Received
September
28,
Bouhours
and Danisle
E. Herriot
H Hopital
Bouhours
69374 LYON CEDEX 2 FRANCE
1978
Summary : The major neutral glycolipids of Guinea Pig fundic epithelium were characterized as glucosylceramide, galactosylceramide, digalactosylceramide and trihexosylceramide. Free ceramide was also found in noticeable amount. Quantitative differences in sphingolipid distribution appeared between each individual gastric cell type : mucous cell, chief cell and oxyntic cell. INTRODUCTION Extensive led
to the
However
concept
adequate
investigate a complex study
on erythrocyte
that cell
tissue.
methods
glycolipid
separation
Guinea
Pig
composition
of gastric
stomach,
cell
procedures
are
of the
method
membranes
of villus isolation
cell
offered
cells,
the chief
(1).
to type
of
the opportunity
Rat
of the
glycolipids
specific
unavailable
and crypt types
cell
often
and separation cell
the mucous
is
individual
has already
of the major
namely
cell
distribution
composition
A convenient
analysis
and cultured
glycosphingolipid
the glycolipid
the glycolipid
Recent the
studies
intestinal (3)
fundic cells
to
cells
portion
of
and the oxyntic
cells. MATERIALS AND METHODS Isolation and separation of gastric cells. The fundic portion of the stomach was cut out of a male Guinea Pig (350 g), thoroughly washed under tap water and opened along the great curvature. The tissue was firmly pinned, mucosa upward, on histology wax previously cast in a glass dish. Epithelial cells were isolated by pronase digestion (4) and separated by velocity sedimentation at unit gravity (3) in three fractions : mucous cells (mean diameter 10.9 p), chief cells (mean diameter 13.7 p) and oxyntic cells (mean diameter 22 p). Lipid extraction. Cell pellets obtained after three washings with buffer were frozen. Analyses were conducted on cells collected from 10 animals. Cells were first homogenized in 7 volumes of methanol then 14 volumes of chloroform were added. After overnight extraction at room temperature, the suspension was centrifuged, the supernatant was collected first with chloroform-methanol and the protein residue was reextracted twice, (l:l), then with (2:l). Lipid extract was concentrated and partitionned according to Folch --et al. (5). GL-2, dihexosylceramide; ABBREVIATIONS : GL-1, monohexosylceramide; GL-3, trihexosylceramide; g.l.c., gas liquid chromatography; t.l.c., thinlayer chromatography; TFA, trifluoroacetate. 0006~291X/78/0854-1314$01.00/0 Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
1314
(2).
have made possible
BIOCHEMICAL
Vol. 85, No. 4, 1978
1 : T.1.c.
Figure
analysis
AND BIOPHYSICAL
of glycolipids
RESEARCH COMMUNICATIONS
from Guinea
Pig
gastric
epithelium.
Visualization by a-naphtol-sulfuric acid sprays. Glycolipids from rat intestine (lane I), from Guinea Pig gastric scraping (lane Z), from Guinea Pig isolated cells before separation in three types (lane 3), from milk fat globule membrane : glucosylceramide (lane 4), lactosylceramide (lane 5).
Glycolipid separation and quantitation. The lipid extracts were applied to a Biosil column (I g). Neutral lipids were eluted with chloroform (ib ml), glycolipids with acetone (15 ml) and acetonelnethanol (9:l) (30 ml). The glycolipid fraction was submitted to a mild alkaline methanolysis (6) and analyzed on precoated thin-layer plates (Merck) in the solvent system chloroform-methanol-water (60:35:8). Sphingosine content of individual glycolipids was determined with Fluorescamine (Roche) (2). Glycolipid identification. Individual glycolipids separated by thinlayer chromatography were methanolyzed at 85'C for 16 hours in anhydrous methanol-HCl 0.75 N. After fatty acid extraction with hexane, the methanol phase was dried under N2 then derivatives were made and analyzed by g.1.c. according to Zanetta --et al. (7). The globoside isolated from human erythrocyte membrane was analyzed and used as reference. RESULTS The glycolipid
composition
established
on a mucosal
Three major characteristics
spots
Under
carbohydrate
ceramide
appeared identical analysis
(56% of GL-1)
of Guinea
scraping
Pig
epithelium
of the fundic
portion
has been of the
stomach.
on thin-layer plates (Fig.1, lane 2) with migration to the reference standards GL-1, GL-2 and GL-3. (Table
I),
there
and a galactosylceramide
1315
were
two GL-1
: a glucosyl-
(44X of GL-1).
GL-2 was
Vol. 85, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE I : Sugars and sphingosine content of individual glycolipids isolated from a mucosal scraping of Guinea Pig fundic epithelium. Glycolipids
Sphingosine
GL-I GL-2 GL-3 GL-4
Glc
Gal
GalNAc
I 1 1 n.d.
0.66 0.12 1.03 1.00
0.52 2.18 2.09 1.90
0.69
I
1.00
1.89
0.78
Human erythrocyte GL-4
Results are expressed as molar ratios of individual sugars to sphingosine GL-I, GL-2, GL-3 and human erythrocyte GL-4, and to glucose for GL-4. Sphingosine was quantified by a fluorimetric method and sugars by g.1.c. analysis of their TPA derivatives. Reference analysis of human erythrocyte globoside was performed with identical procedures.
TABLE II
: Molar
distribution of glycosphingolipids and free in Guinea Pig isolated gastric cells.
Sphingolipids
Isolated mucous
A fourth
spot
containing
was barely
glycolipid
contribution with
and GL-3
(below
accuracy
visible
cell
of the
comparison
a direct
of free
ceramide.
glycolipid, and GL-3 cells
cell
In the
followed (Table
and lower
II).
glycolipids) of
isolated
Furthermore three
GL-3
content
oxyntic
This
% 15 35 46 4
N Acetyl-galactosamine as a globoside.
was too gastric
content
on a molar
low
this
Its
to be determined
was higher cells.
Beside
1316
detected
types,
of magnitude
was established spots.
of glycolipid method
cell
order
cells
of individual
basis
gastric
in decreasing
in the
GL-3. identified
sphingosine
types.
% 28 31 33 8
glycolipids.
composition
by determination the different
below
1% of mucosal
allowed
oxyntic
a galactosyl-galactosyl-glucosylceramide.
was tentatively
in isolated
The glycolipid
ceramide
types
chief
% 28 28 31 13
Free ceramide GL-I GL-2 GL-3
a digalactosylceramide
cell
for
This
patterns
method of
the presence
GL-2 was the major by GL-I,
in mucous cells GL-3 content,
free than
mucous
ceramide in chief and
BIOCHEMICAL
Vol. 85, No. 4, 1978
chief
cell
glycolipids
characterized content.
showed
by the highest GL-1
AND BIOPHYSICAL
only
minor
differences.
GL-2 content
contribution
to the
other
Oxyntic
and the lowest
was the same in the three
The glucosylceramide:galactosylceramide type
RESEARCH COMMUNICATIONS
ratio
as determined
by g.1.c.
free
cell
was not
of glucose
cells
were
ceramide
types.
different
from
one cell
and galactose.
DISCUSSION In our a villus-crypt composition
gradient tissue.
glycolipid the
studies,
the
Gastric
composition
noticeable
the three quantitative
versus cell
The present cell
cell
types
separation
the study allowed
different study have
cells
within
to investigate mature
demonstrates the
according
to
of the glycolipid
undifferentiated
separation
of functionally
same stem cells.
differentiation
intestinal
has made possible
of differentiated
epithelial from
previous
cells that
same glycolipids
a‘normal the
originating after but
with
differences.
ACKNOWLEDGEMENTS : supported by INSERM grant (D. Bouhours) from DGRST nO77.129.
CL.77.5.131.7
and a scholarship
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Hakomori, S.I. (1975) Biochim. Biophys. Acta 417, 55-89. Bouhours, J.F., and Glickman, R.M. (1976) Bioxm. Biophys. Acta 441, 123-133. Romrell, L.J., Coppe, M.R., Munro, D.R., and Ito, S. (1975) J. Cell. Biol. 65, 428-438. Blum, A.L., Shah, G.T., Wiebelhaus, V.D., Brennan, F.T., Helander, H.F., Ceballos, R., and Sachs, G. (1971) Gastroenterology 2, 189-200. Folch, J., Lees, M., and Sloane Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509. Vance, D.E., and Sweeley, C.C. (1967) J. Lip. Res. S, 621-630. Zanetta, J.P., Breckenridge, W.C., and Vincendon, G. (1972) J. Chromatography 69, 291-304.
1317
Vol. 85, No. 4, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
December 29,1978
NEUTRAL GLYCOSPHINGOLIPIDS
1314-1317
OF THREE CELL TYPES ISOLATED FROM GUINEA PIG GASTRIC MUCOSA
Jean-Franfois INSERM U 45 Pavillon Received
September
28,
Bouhours
and Danisle
E. Herriot
H Hopital
Bouhours
69374 LYON CEDEX 2 FRANCE
1978
Summary : The major neutral glycolipids of Guinea Pig fundic epithelium were characterized as glucosylceramide, galactosylceramide, digalactosylceramide and trihexosylceramide. Free ceramide was also found in noticeable amount. Quantitative differences in sphingolipid distribution appeared between each individual gastric cell type : mucous cell, chief cell and oxyntic cell. INTRODUCTION Extensive led
to the
However
concept
adequate
investigate a complex study
on erythrocyte
that cell
tissue.
methods
glycolipid
separation
Guinea
Pig
composition
of gastric
stomach,
cell
procedures
are
of the
method
membranes
of villus isolation
cell
offered
cells,
the chief
(1).
to type
of
the opportunity
Rat
of the
glycolipids
specific
unavailable
and crypt types
cell
often
and separation cell
the mucous
is
individual
has already
of the major
namely
cell
distribution
composition
A convenient
analysis
and cultured
glycosphingolipid
the glycolipid
the glycolipid
Recent the
studies
intestinal (3)
fundic cells
to
cells
portion
of
and the oxyntic
cells. MATERIALS AND METHODS Isolation and separation of gastric cells. The fundic portion of the stomach was cut out of a male Guinea Pig (350 g), thoroughly washed under tap water and opened along the great curvature. The tissue was firmly pinned, mucosa upward, on histology wax previously cast in a glass dish. Epithelial cells were isolated by pronase digestion (4) and separated by velocity sedimentation at unit gravity (3) in three fractions : mucous cells (mean diameter 10.9 p), chief cells (mean diameter 13.7 p) and oxyntic cells (mean diameter 22 p). Lipid extraction. Cell pellets obtained after three washings with buffer were frozen. Analyses were conducted on cells collected from 10 animals. Cells were first homogenized in 7 volumes of methanol then 14 volumes of chloroform were added. After overnight extraction at room temperature, the suspension was centrifuged, the supernatant was collected first with chloroform-methanol and the protein residue was reextracted twice, (l:l), then with (2:l). Lipid extract was concentrated and partitionned according to Folch --et al. (5). GL-2, dihexosylceramide; ABBREVIATIONS : GL-1, monohexosylceramide; GL-3, trihexosylceramide; g.l.c., gas liquid chromatography; t.l.c., thinlayer chromatography; TFA, trifluoroacetate. 0006~291X/78/0854-1314$01.00/0 Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
1314
(2).
have made possible
BIOCHEMICAL
Vol. 85, No. 4, 1978
1 : T.1.c.
Figure
analysis
AND BIOPHYSICAL
of glycolipids
RESEARCH COMMUNICATIONS
from Guinea
Pig
gastric
epithelium.
Visualization by a-naphtol-sulfuric acid sprays. Glycolipids from rat intestine (lane I), from Guinea Pig gastric scraping (lane Z), from Guinea Pig isolated cells before separation in three types (lane 3), from milk fat globule membrane : glucosylceramide (lane 4), lactosylceramide (lane 5).
Glycolipid separation and quantitation. The lipid extracts were applied to a Biosil column (I g). Neutral lipids were eluted with chloroform (ib ml), glycolipids with acetone (15 ml) and acetonelnethanol (9:l) (30 ml). The glycolipid fraction was submitted to a mild alkaline methanolysis (6) and analyzed on precoated thin-layer plates (Merck) in the solvent system chloroform-methanol-water (60:35:8). Sphingosine content of individual glycolipids was determined with Fluorescamine (Roche) (2). Glycolipid identification. Individual glycolipids separated by thinlayer chromatography were methanolyzed at 85'C for 16 hours in anhydrous methanol-HCl 0.75 N. After fatty acid extraction with hexane, the methanol phase was dried under N2 then derivatives were made and analyzed by g.1.c. according to Zanetta --et al. (7). The globoside isolated from human erythrocyte membrane was analyzed and used as reference. RESULTS The glycolipid
composition
established
on a mucosal
Three major characteristics
spots
Under
carbohydrate
ceramide
appeared identical analysis
(56% of GL-1)
of Guinea
scraping
Pig
epithelium
of the fundic
portion
has been of the
stomach.
on thin-layer plates (Fig.1, lane 2) with migration to the reference standards GL-1, GL-2 and GL-3. (Table
I),
there
and a galactosylceramide
1315
were
two GL-1
: a glucosyl-
(44X of GL-1).
GL-2 was
Vol. 85, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE I : Sugars and sphingosine content of individual glycolipids isolated from a mucosal scraping of Guinea Pig fundic epithelium. Glycolipids
Sphingosine
GL-I GL-2 GL-3 GL-4
Glc
Gal
GalNAc
I 1 1 n.d.
0.66 0.12 1.03 1.00
0.52 2.18 2.09 1.90
0.69
I
1.00
1.89
0.78
Human erythrocyte GL-4
Results are expressed as molar ratios of individual sugars to sphingosine GL-I, GL-2, GL-3 and human erythrocyte GL-4, and to glucose for GL-4. Sphingosine was quantified by a fluorimetric method and sugars by g.1.c. analysis of their TPA derivatives. Reference analysis of human erythrocyte globoside was performed with identical procedures.
TABLE II
: Molar
distribution of glycosphingolipids and free in Guinea Pig isolated gastric cells.
Sphingolipids
Isolated mucous
A fourth
spot
containing
was barely
glycolipid
contribution with
and GL-3
(below
accuracy
visible
cell
of the
comparison
a direct
of free
ceramide.
glycolipid, and GL-3 cells
cell
In the
followed (Table
and lower
II).
glycolipids) of
isolated
Furthermore three
GL-3
content
oxyntic
This
% 15 35 46 4
N Acetyl-galactosamine as a globoside.
was too gastric
content
on a molar
low
this
Its
to be determined
was higher cells.
Beside
1316
detected
types,
of magnitude
was established spots.
of glycolipid method
cell
order
cells
of individual
basis
gastric
in decreasing
in the
GL-3. identified
sphingosine
types.
% 28 31 33 8
glycolipids.
composition
by determination the different
below
1% of mucosal
allowed
oxyntic
a galactosyl-galactosyl-glucosylceramide.
was tentatively
in isolated
The glycolipid
ceramide
types
chief
% 28 28 31 13
Free ceramide GL-I GL-2 GL-3
a digalactosylceramide
cell
for
This
patterns
method of
the presence
GL-2 was the major by GL-I,
in mucous cells GL-3 content,
free than
mucous
ceramide in chief and
BIOCHEMICAL
Vol. 85, No. 4, 1978
chief
cell
glycolipids
characterized content.
showed
by the highest GL-1
AND BIOPHYSICAL
only
minor
differences.
GL-2 content
contribution
to the
other
Oxyntic
and the lowest
was the same in the three
The glucosylceramide:galactosylceramide type
RESEARCH COMMUNICATIONS
ratio
as determined
by g.1.c.
free
cell
was not
of glucose
cells
were
ceramide
types.
different
from
one cell
and galactose.
DISCUSSION In our a villus-crypt composition
gradient tissue.
glycolipid the
studies,
the
Gastric
composition
noticeable
the three quantitative
versus cell
The present cell
cell
types
separation
the study allowed
different study have
cells
within
to investigate mature
demonstrates the
according
to
of the glycolipid
undifferentiated
separation
of functionally
same stem cells.
differentiation
intestinal
has made possible
of differentiated
epithelial from
previous
cells that
same glycolipids
a‘normal the
originating after but
with
differences.
ACKNOWLEDGEMENTS : supported by INSERM grant (D. Bouhours) from DGRST nO77.129.
CL.77.5.131.7
and a scholarship
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Hakomori, S.I. (1975) Biochim. Biophys. Acta 417, 55-89. Bouhours, J.F., and Glickman, R.M. (1976) Bioxm. Biophys. Acta 441, 123-133. Romrell, L.J., Coppe, M.R., Munro, D.R., and Ito, S. (1975) J. Cell. Biol. 65, 428-438. Blum, A.L., Shah, G.T., Wiebelhaus, V.D., Brennan, F.T., Helander, H.F., Ceballos, R., and Sachs, G. (1971) Gastroenterology 2, 189-200. Folch, J., Lees, M., and Sloane Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509. Vance, D.E., and Sweeley, C.C. (1967) J. Lip. Res. S, 621-630. Zanetta, J.P., Breckenridge, W.C., and Vincendon, G. (1972) J. Chromatography 69, 291-304.
1317
