17.
A C T I O N S O F
V I T A M I N S
A
A N D
C
687
the D K G stage (Rinaldini, unpublished). This shows that exogenous
ASA is not required for contraction, although it is normally present
in muscle cells. Chick embryonic muscle contains from 100 to 200 /xg
ASA/g (wet weight) depending on the age and, if credence can be
given to histochemical reduction tests, the vitamin appears in myoblasts before any sign of anatomical differentiation (Weel, 1948).
Yet muscle is unable to synthesize ASA (Fabro and Rinaldini, unpublished) and consequently one would expect its reserves to be soon
depleted in an ASA-free medium. One is justified in concluding,
therefore, that the role of ASA in muscle cells is not concerned with
contraction.
In a series of unpublished experiments on cultures of freshly isolated
chick embryonic myoblasts I was surprised to find that ASA added
to a basal non-growth-promoting medium in concentrations of 100-200
/xg/ml, which are ten to twenty times higher than those in plasma but
are equivalent to those found in embryonic muscle of the same age
in vivo, had a strong inhibitory effect on cell spreading and survival,
suggesting an accumulation in the cell. Inhibition was almost complete
with 300 /xg/ml. If the ASA was added after the cells were fully spread
it had at first no apparent effect on their morphology, but if the dosage
was repeated daily they eventually became dark and shrunken and
died within a week. A delayed effect was observed in one set of cultures
where a single dose caused severe degeneration 4 days after being
administered.
D H A at the same concentration had no effect, indicating that the
dienol group of ASA is necessary for activity. This suggested that the
cytotoxic effect might be due to the reducing action, particularly since
Havard and Kendall (1934) had shown that mitosis and migration
of cells in explant cultures of chick heart were inhibited by lowering
the redox potential and that they ceased almost entirely at an Eh of
-0-03 v. Green (1933) estimated the redox potential of ASA at pH 7-08
and 30°C to be - 5 4 mv at 100% reduction and Ball (1937) found an
E'o of +133-4 mv. The relatively small doses added to the cultures,
however, would result in a low poising effect. Moreover, equivalent
doses of GSH, which has a stronger reducing power than ASA, left
the cells completely unaffected. The explanation of the inhibitory
effect must, therefore, be sought in other properties of ASA, and it may
be connected with its curious lytic effect on intercellular mucins
discussed below. This would fit with the observation that its action was
most marked when added to the cells before they were allowed to
spread, and while they were engaged in the process of rebuilding their
own extracellular coatings.
The concentrations used in these experiments were of the order of
A C T I O N S O F
V I T A M I N S
A
A N D
C
687
the D K G stage (Rinaldini, unpublished). This shows that exogenous
ASA is not required for contraction, although it is normally present
in muscle cells. Chick embryonic muscle contains from 100 to 200 /xg
ASA/g (wet weight) depending on the age and, if credence can be
given to histochemical reduction tests, the vitamin appears in myoblasts before any sign of anatomical differentiation (Weel, 1948).
Yet muscle is unable to synthesize ASA (Fabro and Rinaldini, unpublished) and consequently one would expect its reserves to be soon
depleted in an ASA-free medium. One is justified in concluding,
therefore, that the role of ASA in muscle cells is not concerned with
contraction.
In a series of unpublished experiments on cultures of freshly isolated
chick embryonic myoblasts I was surprised to find that ASA added
to a basal non-growth-promoting medium in concentrations of 100-200
/xg/ml, which are ten to twenty times higher than those in plasma but
are equivalent to those found in embryonic muscle of the same age
in vivo, had a strong inhibitory effect on cell spreading and survival,
suggesting an accumulation in the cell. Inhibition was almost complete
with 300 /xg/ml. If the ASA was added after the cells were fully spread
it had at first no apparent effect on their morphology, but if the dosage
was repeated daily they eventually became dark and shrunken and
died within a week. A delayed effect was observed in one set of cultures
where a single dose caused severe degeneration 4 days after being
administered.
D H A at the same concentration had no effect, indicating that the
dienol group of ASA is necessary for activity. This suggested that the
cytotoxic effect might be due to the reducing action, particularly since
Havard and Kendall (1934) had shown that mitosis and migration
of cells in explant cultures of chick heart were inhibited by lowering
the redox potential and that they ceased almost entirely at an Eh of
-0-03 v. Green (1933) estimated the redox potential of ASA at pH 7-08
and 30°C to be - 5 4 mv at 100% reduction and Ball (1937) found an
E'o of +133-4 mv. The relatively small doses added to the cultures,
however, would result in a low poising effect. Moreover, equivalent
doses of GSH, which has a stronger reducing power than ASA, left
the cells completely unaffected. The explanation of the inhibitory
effect must, therefore, be sought in other properties of ASA, and it may
be connected with its curious lytic effect on intercellular mucins
discussed below. This would fit with the observation that its action was
most marked when added to the cells before they were allowed to
spread, and while they were engaged in the process of rebuilding their
own extracellular coatings.
The concentrations used in these experiments were of the order of