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Cystic fibrosis: The effect of medium from cultured cystic fibrosis fibroblasts on ATPase activity
183
Clinica Chimica Acta, 74 (1977) 183-185 @ ElsevieriNorth-Holland Biomedical Press
SHORT COMMUNICATION _._ .._ ~~~~_~ ~~ CCA 8262
CYSTIC FIBROSIS: THE EFFECT OF MEDIUM FROM CULTURED FIBROSIS FIBROBLASTS ON ATPase ACTIVITY
D.A. APPLEGARTH *, A.G.F. DAVIDSON, ROUFOGALIS and D.G. CLARK
E.M. HAWORTH,
Departments of Paediatrics, Pharmaceutical Sciences British Colum biu, Vancouver, B.C. (Canada) (Received
July 26th,
E.E. QUIST,
and Chemistry,
CYSTIC
R. ZUK, B.D.
The University
of
19 16)
Summary Culture medium from ‘fibroblasts of cystic fibrosis patients and controls was examined for the ability to inhibit (calcium plus magnesium)-activated ATPase and (sodium plus potassium)-activated ATPase. The ATPase systems used were both a solubilised preparation from dog-fish and a membrane associated preparation from human erythrocytes. Contrary to other reports the medium from cystic fibrosis fibroblasts did not inhibit ATPase activity. Introduction Culture medium from fibroblasts of cystic fibrosis patients has been reported to have an inhibitory effect on calcium and sodium plus potassium activated ATPase activities of human red cells [ 11. As a first step towards examining whether this phenomenon could explain the “ciliary dyskinesia” or other effects reported to occur in cystic fibrosis, we have re-examined the effect of medium from cultured cystic fibrosis fibroblasts on human red cell ATPase activity. Our findings show no difference between cystic fibrosis and control cell lines with respect to the ability of their spent culture medium to affect human red cell ATPase activity. Methods
and materials
Fibroblasts from 4-mm biopsies of skin obtained from the deltoid area were cultured in Eagles’ minimum essential medium with Earle’s salts, 10% fetal calf * Reprint
requests
Laboratory.
should
Children’s
be addressed Hospital,
250
to: West
Dr.
Derek
59th
A.
Avenue,
Applegarth, Vancouver,
Biochemical B.C.
V5X
Diseases 1X2,
Canada.
serum, nonessential amino acids and 100 I.U./ml penicillin and streptomycin, using disposable plastic culture flasks. Cell lines were taken through at least 4 generations before use. Immediately after confluency, the medium was transferred to plastic beakers and dialyzed against running tap water overnight using a membrane with a molecular weight cut off of 3500 (Spectropore 3, Fisher Scientific). Material remaining in the dialysis sac was freezedried and reconstituted in water. An aliquot corresponding to approximately 1 mg of protein was added to the assay system to measure ATPase activities according to the method of Quist and Roufogalis [ 21, using erythrocyte membrane preparation A. We also evaluated the effect of the culture medium on a solubilised ATPase system prepared from the rectal gland of a dog-fish by the method of Hokin et al. [ 31. An aliquot of fibroblast culture medium corresponding to approximately 0.5 mg of protein was added to the assay system, which consisted of 30 mM imidazolc, 120 mM sodium chloride, 7 mM magnesium chloride, 20 mM potassium chloride, 0.006% phosphatidyl serine and 4 mM ATP, at a pH of 6.5.
,\TPasr
ACTIVITIES
TCTRE
Ml?DItiM
A.
+ Ca)-ATPase
(Mg
Assays
were
OF FROM
I
MEMBRANES
FIBROBLASTS
activity
performed
with
OF
cxpresscd 0.2
mM
as nmol CaCll
MEASURED
PATIENTS
AND
inorganic
and
0.1
IN
phosphate/rug
mM
Ouabain. medium
Controls
Patients
Unused
333
336
283
301
308
509
524
THE
PRESI
OF
CUL-
CONTROLS protein/h.
All
other
conditions
as in reference
2.
509 589 320 377,411 480 hlean ____
= 416
r 111
Mean __~_~
____
B. (Na
+ K)-?\TPase
Assavs
were
difference in reference
activity
performed between
expressed
without the
= 389
as nmol
added
activitirs
in the
CaCI2, absence
inorganic with
phosphateimg
66
and
mM
presence
Patients
IJnused 126
179
201
151
160
144
129
122 135 113.126 138,116 = 140
of
2
Controls
Mean
NaCI
t
21
Mean
= 163
medium
and 0.1
protein/h. 10 mM
mM
KCI.
ouabain.
Results All
other
represent conditions
the as
185
TABLE
II
ATPasc
ACTIVITIISS
ENCE
OF
OF
CULTllRE
A. Mg-ATPase
activity
LUBROL
W’X
~~~DIU~~
FROM
expressed
as nmol
SOLUBlLISED
inorganic
Patients
Unused 17
22
24 22
12
16
OF
ATPasc
PATIENT
phosphate/mg
Controls
28
DOG-FISH
FIBROBLASTS
AND
MEASURED
IN
THE
PRES-
CONTROLS
protein/h.
medium
8 32 Mean
Mean
= 20.4
B. (Na
+ KkATPase
Assays
were
swine
= 20.3
activity
performed
as dcsrribed
expressed
in
imida~ol~/Ii~I
buffer,
pFi
7.0,
Unused
medium
90
88
85
92
97
90
76
mM
protein/h.
containing
Patients
Controls
op0.1
srction.
phosphatc/mg
represent
and absence _. _.-.-
methods
inorganic
Results
presence
in the
as nmol
the
essential
difference
cations
between
and tbr
phos~batid~l
activities
in the
ouabain. -.-
94 100 79 80 Mean
Mean
= 89.3
= 87
Results Table I shows the membrane associated ATPase activity of human red blood membranes assayed with and without addition of the dialyzed medium. Table II shows the effect of the dialyzed medium on the solubilised ATPase system. Discussion We found no inhibition activated ATPase activities sis patients. This was true red blood cell membranes in accepting the conclusion quality of culture medium
of magnesium plus calcium or sodium plus potassium by culture medium of fibrobl~~ from cystic fibrowhether the ATPase used was obtained from human or dog-fish rectal glands. Our results suggest caution that inhibition of ATPase activity is a characteristic from fibroblasts of cystic fibrosis patients.
References 1
Schmoyer,
I.R.
and
2
Quist,
and
Roufogalis,
3
Hokin, Chem.
4
E.E.
See,
L.E..
Dahl,
Baglia, J.L.,
F.A.
(1974)
B.D.
Biochem.
(1975)
Doupree.
J.D.,
Arch.
Biophys.
Biochem.
Dixon,
J.F..
248.2593 Y.P.
and
Fitt,
P.S.
(1972)
Anal.
Biochem.
49.
430
Res. Biophys.
Hackeny,
Commun. 168, J.F.
58,
1066
240 and
Perdue,
J.F.
(1975)
J.
Biol.
Clinica Chimica Acta, 74 (1977) 183-185 @ ElsevieriNorth-Holland Biomedical Press
SHORT COMMUNICATION _._ .._ ~~~~_~ ~~ CCA 8262
CYSTIC FIBROSIS: THE EFFECT OF MEDIUM FROM CULTURED FIBROSIS FIBROBLASTS ON ATPase ACTIVITY
D.A. APPLEGARTH *, A.G.F. DAVIDSON, ROUFOGALIS and D.G. CLARK
E.M. HAWORTH,
Departments of Paediatrics, Pharmaceutical Sciences British Colum biu, Vancouver, B.C. (Canada) (Received
July 26th,
E.E. QUIST,
and Chemistry,
CYSTIC
R. ZUK, B.D.
The University
of
19 16)
Summary Culture medium from ‘fibroblasts of cystic fibrosis patients and controls was examined for the ability to inhibit (calcium plus magnesium)-activated ATPase and (sodium plus potassium)-activated ATPase. The ATPase systems used were both a solubilised preparation from dog-fish and a membrane associated preparation from human erythrocytes. Contrary to other reports the medium from cystic fibrosis fibroblasts did not inhibit ATPase activity. Introduction Culture medium from fibroblasts of cystic fibrosis patients has been reported to have an inhibitory effect on calcium and sodium plus potassium activated ATPase activities of human red cells [ 11. As a first step towards examining whether this phenomenon could explain the “ciliary dyskinesia” or other effects reported to occur in cystic fibrosis, we have re-examined the effect of medium from cultured cystic fibrosis fibroblasts on human red cell ATPase activity. Our findings show no difference between cystic fibrosis and control cell lines with respect to the ability of their spent culture medium to affect human red cell ATPase activity. Methods
and materials
Fibroblasts from 4-mm biopsies of skin obtained from the deltoid area were cultured in Eagles’ minimum essential medium with Earle’s salts, 10% fetal calf * Reprint
requests
Laboratory.
should
Children’s
be addressed Hospital,
250
to: West
Dr.
Derek
59th
A.
Avenue,
Applegarth, Vancouver,
Biochemical B.C.
V5X
Diseases 1X2,
Canada.
serum, nonessential amino acids and 100 I.U./ml penicillin and streptomycin, using disposable plastic culture flasks. Cell lines were taken through at least 4 generations before use. Immediately after confluency, the medium was transferred to plastic beakers and dialyzed against running tap water overnight using a membrane with a molecular weight cut off of 3500 (Spectropore 3, Fisher Scientific). Material remaining in the dialysis sac was freezedried and reconstituted in water. An aliquot corresponding to approximately 1 mg of protein was added to the assay system to measure ATPase activities according to the method of Quist and Roufogalis [ 21, using erythrocyte membrane preparation A. We also evaluated the effect of the culture medium on a solubilised ATPase system prepared from the rectal gland of a dog-fish by the method of Hokin et al. [ 31. An aliquot of fibroblast culture medium corresponding to approximately 0.5 mg of protein was added to the assay system, which consisted of 30 mM imidazolc, 120 mM sodium chloride, 7 mM magnesium chloride, 20 mM potassium chloride, 0.006% phosphatidyl serine and 4 mM ATP, at a pH of 6.5.
,\TPasr
ACTIVITIES
TCTRE
Ml?DItiM
A.
+ Ca)-ATPase
(Mg
Assays
were
OF FROM
I
MEMBRANES
FIBROBLASTS
activity
performed
with
OF
cxpresscd 0.2
mM
as nmol CaCll
MEASURED
PATIENTS
AND
inorganic
and
0.1
IN
phosphate/rug
mM
Ouabain. medium
Controls
Patients
Unused
333
336
283
301
308
509
524
THE
PRESI
OF
CUL-
CONTROLS protein/h.
All
other
conditions
as in reference
2.
509 589 320 377,411 480 hlean ____
= 416
r 111
Mean __~_~
____
B. (Na
+ K)-?\TPase
Assavs
were
difference in reference
activity
performed between
expressed
without the
= 389
as nmol
added
activitirs
in the
CaCI2, absence
inorganic with
phosphateimg
66
and
mM
presence
Patients
IJnused 126
179
201
151
160
144
129
122 135 113.126 138,116 = 140
of
2
Controls
Mean
NaCI
t
21
Mean
= 163
medium
and 0.1
protein/h. 10 mM
mM
KCI.
ouabain.
Results All
other
represent conditions
the as
185
TABLE
II
ATPasc
ACTIVITIISS
ENCE
OF
OF
CULTllRE
A. Mg-ATPase
activity
LUBROL
W’X
~~~DIU~~
FROM
expressed
as nmol
SOLUBlLISED
inorganic
Patients
Unused 17
22
24 22
12
16
OF
ATPasc
PATIENT
phosphate/mg
Controls
28
DOG-FISH
FIBROBLASTS
AND
MEASURED
IN
THE
PRES-
CONTROLS
protein/h.
medium
8 32 Mean
Mean
= 20.4
B. (Na
+ KkATPase
Assays
were
swine
= 20.3
activity
performed
as dcsrribed
expressed
in
imida~ol~/Ii~I
buffer,
pFi
7.0,
Unused
medium
90
88
85
92
97
90
76
mM
protein/h.
containing
Patients
Controls
op0.1
srction.
phosphatc/mg
represent
and absence _. _.-.-
methods
inorganic
Results
presence
in the
as nmol
the
essential
difference
cations
between
and tbr
phos~batid~l
activities
in the
ouabain. -.-
94 100 79 80 Mean
Mean
= 89.3
= 87
Results Table I shows the membrane associated ATPase activity of human red blood membranes assayed with and without addition of the dialyzed medium. Table II shows the effect of the dialyzed medium on the solubilised ATPase system. Discussion We found no inhibition activated ATPase activities sis patients. This was true red blood cell membranes in accepting the conclusion quality of culture medium
of magnesium plus calcium or sodium plus potassium by culture medium of fibrobl~~ from cystic fibrowhether the ATPase used was obtained from human or dog-fish rectal glands. Our results suggest caution that inhibition of ATPase activity is a characteristic from fibroblasts of cystic fibrosis patients.
References 1
Schmoyer,
I.R.
and
2
Quist,
and
Roufogalis,
3
Hokin, Chem.
4
E.E.
See,
L.E..
Dahl,
Baglia, J.L.,
F.A.
(1974)
B.D.
Biochem.
(1975)
Doupree.
J.D.,
Arch.
Biophys.
Biochem.
Dixon,
J.F..
248.2593 Y.P.
and
Fitt,
P.S.
(1972)
Anal.
Biochem.
49.
430
Res. Biophys.
Hackeny,
Commun. 168, J.F.
58,
1066
240 and
Perdue,
J.F.
(1975)
J.
Biol.