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The effect of vitamin A, hydrocortisone and citral upon sulphate metabolism in skin
Experimental
Cell Research
35, 255-261
(1964)
255
THE EFFECT OF VITAMIN A, HYDROCORTISONE AND CITRAL UPON SULPHATE METABOLISM IN SKIN S. A. BARKER, Medical
C. N. D. CRUICKSHANK
Research Council, Unit for Research the Department of Chemistry, University Received
on the Experimental of Birmingham,
and TESSA Pathology Birmingham,
WEBB of the Skin England
and
July 23, 1963
IN 1953 Fell and Melanby [6] made the fundamental discovery that the administration of vitamin A to tissue cultures of chicken embryo skin caused the normal stratified squamous epithelium to undergo metaplasia to a mucous secreting epithelium and that this change was associated with the accumulation of mucopolysaccharide in the epidermal cells. Pelt and Fell [ 121 demonstrated autoradiographically an increased incorporation of 35Slabelled sulphate into chicken embryo skin under the influence of vitamin A. Lawrence and Ricketts [9] had previously studied the uptake of 35S sulphate by guinea pig skin respiring in vitro, and shown that it could be influenced by heating, bacterial toxins, cortisone or iodoacetic acid. On this basis they concluded that sulphate incorporation was an active enzymatic process and that it was being used for the synthesis of sulphated mucopolysaccharides. Initial studies in this laboratory of organ cultures of adult guinea pig ear skin growing with and without vitamin A, failed to demonstrate any histological effect. Since this might have been due to the fact that the turnover of mucopolysaccharides was likely to be slower in adult than in chicken embryo skin or that the extent to which the adult epidermal cell was capable of undergoing metaplasia was limited, experiments were devised to determine whether any evidence of an effect by vitamin A upon sulphate incorporation could be shown. MATERIALS
AND METHODS
Skin slices were cut free-hand from the dorsum of the guinea pig ear, and floated for 24 hr at 37°C upon the surface of 1 ml of medium containing 0.05 mc Na,%O, added to horse serum 40 per cent and Hanks’ BSS 60 per cent, in a gas phase of 95 per cent oxygen and 5 per cent carbon dioxide. Thin slicesof human skin obtained from excessmaterial used at grafting operations were treated similarly. On removal from the medium, the skin slices were plunged into liquid nitrogen for 30 min, and then dialyzed against three changes of 0.1 M sodium sulphate over 24 hr to remove any surface adsorbed radioactive sulphate. The skin was then rinsed in distilled water and dried to constant weight. Complete destruction of the skin Experimental
Cell
Research
35
256
S. A. Barker,
C. N. D.
Cruickshank
and
Tessa
Webb
fragments in 1 ml of 12 N hydrochloric acid at 100°C was followed by precipitation of the sulphate by addition of 0.1 M sodium sulphate (4 ml) as carrier, followed by 0.1 M barium chloride (5 ml). The solid barium sulphate wasthen placed as an acetone suspensionon to an aluminium planchette. Counting was by the end window method, at the calculated infinite thickness. In someexperiments the epidermis was separated from the dermis after incubation with 0.25 per cent trypsin for 30 min or longer if necessary,and the two tissuestreated separately as above. Further portions of skin were subjected to an extraction procedure to demonstrate the degree of incorporation of the sulphate into mucopolysaccharide. Weighed whole skin was cut into small pieces and extracted with acetone to give a fat fraction. The ground residue was suspended in 0.02 M acetate buffer pH 6, treated with ficin (IO mg) in the presence of L-cysteine as activator and placed upon a slow shaker at 37°C for 24 hr. The digest obtained was extracted three times with a chloroformamyl alcohol mixture (10 : 1 v/v). Protein I was recovered from the organic phase. Addition of 70 per cent perchloric acid (1 ml) to the aqueous phase (20 ml) at 4°C for 30 min gave a precipitate designated protein II. After dialysis of the supernatant, freeze drying the centrifuged solution gave a crude mucopolysaccharide fraction, as shown by staining with alcian blue [8] after separation by paper chromatography in 0.1 M borate buffer pH 10 : ethanol (65 : 35 v/v) or paper electrophoresis in 0.2 M formate buffer pH 2. Acid hydrolysates of the fraction gave positive tests for uranic acid [4] and hexosamine [15]. The specific activity of each fraction was measured exactly as for whole skin. RESULTS
Table I demonstrates that the majority (over 60 per cent) of the sulphate is incorporated into mucopolysaccharide, and that the presence of vitamin A 10 IU/ml= 10-S M, almost doubles the specific activity of the mucopolyTABLE
I. The incorporation of 35S-sulphate into various skin fractions (counts/g/set X 103). Specificactivity Fraction
Vitamin A
Control
4.5 4.9 0.35 0.34 0.23 0.28 0.53 0.83
5.8 7.8 0.46 0.32 0.24 0.31 0.37 0.36
(1) Fat
Experimental
(2)
Protein I
(3)
Protein II
(4)
Mucopolysaccharide
Cell Research
35
% Distribution of activity 19 8 12 61
Sulphafe
mefabolism
in skin
saccharide. Fat appears to have a high specific activity which is lowered by the presence of the vitamin. The amount of fat present, however, constitutes such a small proportion (N 1 per cent) of the whole, that any surface adsorbed 35SOi- ion would considerably magnify the value obtained as specific activity. Table II shows that there is a significantly increased incorporation of sulphate into whole skin slices of guinea pig ear in the presence of vitamin A. It also demonstrates that citral 10-E M has no effect upon the mucopolysaccharides of whole skin and inhibits the vitamin A effect. when the epidermis A considerably different picture emerges, however, and dermis are considered separately. Tables III and IV shorn that for guinea pig and human skin respectively the vitamin A effect is confined entirely to the epidermis and that its presence makes no difyerence to the sulphate TABLE
II.
The effects of vitamin A and citral on whole guinea pig ear (countslglsec X 103).
Experiment No.
A
Control
Citral
Vitamin A and citral
1 2
7.33 f 0.76 5.28 I? 0.82
4.36 i 0.20 3.69k0.16
-
-
3 4
5.94 Yc 0.87 5.20 k 0.10
3.40 F 0.41 2.72 zk 0.09
4.09 k 0.46 2.31 f 0.63
4.12 + 0.12 2.25 IL 0.15
Each
TABLE
Vitamin
skin slices of
III.
value
is mean
f S.E. of three
The effects of vitamin A and citral on guinea and dermis (countslglsec x 103).
Experiment
No.
Vitamin
1 Epidermis Dermis 2 Epidermis Dermis 3 Epidermis Dermis 4 Epidermis Dermis Mean
observations.
of paired
0.78 5.9 1.32 2.5 1.07 3.2 2.1 7.4
A
Control
Citral
0.31 5.5 0.68 2.8
-
0.80 3.6 1.3 5.3
0.66 4.1 1.2 8.3
pig
epidermis
Vitamin A and citral 0.96 3.3 2.3 6.9
observations. Experimental
Cell
Research
35
S. A. Barker,
258
C. N. D. Cruickshank
and Tessa Webb
incorporation of the dermis. Citral, on the other hand, causes a slight increase of sulphate incorporation into dermal polysaccharides, but has no effect on that in the epidermis, which is increased as usual in the presence of vitamin A. Table V shows that hydrocortisone 10-S M has two separate effects, the first to decrease sulphate incorporation into the dermis and the second to act as a IV.
TABLE
The efects of vitamin A and citral on human epidermis and dermis (countslglsec x 103).
Experiment
No.
Vitamin
1 Epidermis Dermis 2 Epidermis Dermis 3 Epidermis Dermis Mean
A
Control
0.22 0.21 4.4 2.7 10.4 1.6
of paired
Citral
Vitamin A and citral
0.14 0.19
-
-
3.0 2.9 8.1 1.6
2.3 3.8 7.9 2.1
2.3 3.8 9.4 1.5
observations.
direct inhibitor of the vitamin A effect upon the epidermis. That these alterations are specific is indicated in Table VI by the absence of any effect by stilboestrol. DISCUSSION
The results obtained in the present study indicate that although no histological changes are detectable in adult skin treated in vitro for a short period V. The effects of vitamin A and hydrocortisone on human epidermis and dermis (counts/g/set X 103).
TABLE
Experiment No. 1 Epidermis Dermis 2 Epidermis Dermis 3 Epidermis Dermis Mean Experimental
Vitamin
Control
Hydrocortisone
9.3 2 0.2 1.4 + 0.4 16.1+ 2.8 6.0 5 0.2 10.4iO.l l.SkO.4
7.8 i: 0.3 1.4kO.2 11.5F0.8 4.8 k 0.5 S.Oi;O.5 1.6k0.2
8.2 * 0.4 0.6kO.l 11.3 k 2.9 2.4 + 0.5 7.8 f 0.6 1.0+0.1
+ S.E. of three Cell Research
observations. 35
Vitamin A + hydrocortisone 7.7 k 0.5 0.6 + 0.0 11.4i:o.5 2.7kO.l 7.2 i. 0.4 1.1 io.1
Sulphate
metabolism
in skin
with excess vitamin A, considerable changes in caused. The increased incorporation of 35S sulphate makes it likely that more polysaccharide is actually evidence for this is at present insufficient. It is of the effect of vitamin A is confined to the epidermis, TABLE
Experiment
Guinea
pig
Human
(1)
Human
(2)
Each
value
VI.
259
the metabolism may be into mucopolysaccharide being produced but the considerable interest that although the same muco-
The effects of vitamin A and stilboestrol on human and guinea pig skin (countslglsec X 103). No.
Vitamin 7.0 k 0.7 9.3kO.l 1.4 IL 0.4 29.5 I! 2.4 4.5 IO.2
(1)
Epidermis Dermis Epidermis Dermis is mean
A
&SE.
of three
Control 3.1+ 0.54 7.8 2 0.7 1.4f0.2 22.6 k 0.6 4.8 i 0.8
Stilboestrol
Vitamin A+ stilboestrol
3.1+ 0.3 s.1* 0.4 1.1 kO.01 19.Ok2.0 5.4 k 0.4
6.6 I!I 0.47 8.5 * 0.1 1.2kO.l 23.5 f 0.6 5.8 t 0.1
observations.
polysaccharides appear to be present in both tissues. This implies that its action is mediated by the epidermal cells and is not directly upon the enzymes involved in sulphation. The apparently double effect of hydrocortisone would appear also to be of some importance, for although it inhibits the increased sulphate uptake caused by vitamin A in the epidermis, it independently lowers the uptake of sulphate in the dermis but not the epidermis of non-vitamin A treated skin. The ability of citral to have an independent effect, namely to increase the uptake of dermal sulphate would seem worthy of further investigation. Citral, a terpcne, has a similar structure to that of part of the vitamin A molecule, and the question arises as to whether further allied compounds might also influence sulphate uptake. There are no comparable studies of sulphate metabolism in relation to the polysaccharides of skin, but the effects of vitamin A upon 35SOi- metabolism in rat colon have been studied. Wolf and Varandani [16] have shown that the lowered incorporation in vitro of inorganic 35S sulphate into colon segments obtained from vitamin A deficient rats can be compensated for by the addition of vitamin A. Moretti and Wolf [ 111have shown vitamin A deficiency in rats causes a lowering of the amount of bound hexosamine in colon segments. Later work on pig colon mucosa [171 implicated a protein fraction precipiExperimental
Cell Research
35
260
S. A. Barker,
C. N. D. Cruickshank
and Tessa Webb
tating at pH 5.2 in this effect on mucopolysaccharide metabolism and the fraction was used to demonstrate 353’-phosphoadenosine-5’-phosphosulphate production, and transfer of 35S sulphate to p-nitrophenol. These workers also found that pre-incubation of the pH 5 enzyme fraction with lipoxidase lowered the overall mucopolysaccharide synthesis, but did not affect the transfer mechanism, indicating that vitamin A has an effect upon the initial activation system of 35S sulphate, as follows: ATP+S Adenosine-5’-phosphosulphate + ATP 4 3’-phosphoadenosine-5’-phosphosulphate
+ pyrophosphate
+ ADP
Vitamin A also has an effect upon the liberation of hydrolytic enzymes contained in a latent state within the lysosome particles of tissues. Such particles generally contain proteases, enzymes able to degrade mucopolysaccharides such as sulphatases, glucosaminidases and glucuronidases as well as nucleases and acid phosphatases [2, 31. Generally, all such enzymes are spilled out almost simultaneously by reagents such as digitonin, detergents and freezing and thawing. Furthermore, vitamin A has also been found to cause the liberation of a protease from the lysosomes of chick cartilage [5]. It is this effect of vitamin A upon the lysosomes which is believed to be inhibited by cortisone and hydrocortisone [a]. This is supported by the work of Fell and Thomas [7] who found that hydrocortisone inhibited the in uitro dissolution of cartilaginous long bone rudiments of chick embryo brought about by organ culture with excess of vitamin A. Apart from this, hydrocortisone causes structural alterations in mast cells [ 11, while its local application on to experimental wounds in rabbit skin, in some cases diminished ground substance metachromasia, degranulated the mast cells and reduced their number [18]. Other workers [IO] have shown that a concentration of hydrocortisone in excess of 0.1 mg/ml of medium suppresses the fixation of labelled sulphate by heart and skeletal muscle. The same workers, however, found that similar concentrations of the steroid had no effect upon 35S sulphate incorporation by liver, and expressed the belief that this indicated a different mechanism. Little is known of the metabolic function of citral, save that it has been found not to be a vitamin A inhibitor in cases of glaucoma [14]. At first glance this does not appear to be true of skin, but closer inspection reveals that while Experimental
Cell Research
35
Sulphate
metabolism
in skin
the vitamin A effect lay in the epidermis, the effect of citral was apparently independent as it lay in the dermis only. It remains to be discovered whether there is any relationship between the two. Although oestradiol has been reported as inhibiting 35S sulphate incorporation into tissue [13], we found stilboestrol to have no effect. SUMMARY
I. Over 60 per cent of the 35S sulphate taken up by skin was shown to be contained in a mucopolysaccharide fraction. 2. Vitamin A caused the specific activity of the mucopolysaccharide fraction to be nearly doubled. 35S sulphate incorporation lay 3. The ability of vitamin A to increase entirely in the epidermis. 1. Hydrocortisone, while inhibiting the vitamin A effect in the epidermis, also caused the 35S sulphate uptake in the dermis to be lowered. We are grateful for the invaluable technical assistance of Miss Gabriel West-Samuel. One of us (T.W.) is in receipt of a University of Birmingham Scholarship. REFERENCES 1. ASBOE HANSEN, C., PhysioZ. Reu. 38, 446 (1958). 2. DE DUVE, C., Biochem. Pharmacol. 9, 97 (1962). 3. DINGLE, J. T., Biochem. J. 79, 509 (1961). 4. DISCHE, Z., J. Mol. Chem. 167, 189 (1947). 5. FELL, H. B. and DIPI’GLE, J. T., Biochem. J. 87, 403 (1963). 6. FELL, H. B. and MELANBY, E., J. Physiol. 119, 470 (1953). 7. FELL, H. B. and THOMAS, J., J. ExptZ Med. 114, 343 (1961). 8. HEREMANS, J. and VAERMAN, J. P., Clin. Chim. Acta 3, 430 (1958). 9. LAWRENCE, J. C. and RICKETTS, C. R., Exptl Cell Res. 12, 633 (1957). 10. LAYTON, L., Proc. Sot. Exptl BioZ. Med. 76, 596 (1951). 11. MORETTI, A. and WOLF, G., Biochim. Biophys. Acla 53, 263 (1961). 12. PELC, S. R. and FELL, H. B., ExptZ Cell Res. 19, 99 (1960). 13. PRIEST, R. E. and KOPLITZ, R. M., J. ExptZ Med. 116, 565 (1962). 14. REDGER, F., SCUDUZZAFAR, M. and DUYAL, Y., Trans. Ophthal. Sot. 79, 283 (1959). 15. RONDLE, C. and MORGAN, M., Biochem. J. 61, 586 (1955). 16. WOLF, G. and VARANDANI, P., Biochim. Biophys. Acta 43, 501 (1960). 17. WOLF, G., VARANDANI, P. and JOHNSON, B. C., Riochim. Biophys. Acta, 46, 59 (1961). 18. ZACHARIAE, L. and MOLTKE, E., Acta Endocrinol. 16, 300 (1954).
Experimental
Cell
Research
35
Cell Research
35, 255-261
(1964)
255
THE EFFECT OF VITAMIN A, HYDROCORTISONE AND CITRAL UPON SULPHATE METABOLISM IN SKIN S. A. BARKER, Medical
C. N. D. CRUICKSHANK
Research Council, Unit for Research the Department of Chemistry, University Received
on the Experimental of Birmingham,
and TESSA Pathology Birmingham,
WEBB of the Skin England
and
July 23, 1963
IN 1953 Fell and Melanby [6] made the fundamental discovery that the administration of vitamin A to tissue cultures of chicken embryo skin caused the normal stratified squamous epithelium to undergo metaplasia to a mucous secreting epithelium and that this change was associated with the accumulation of mucopolysaccharide in the epidermal cells. Pelt and Fell [ 121 demonstrated autoradiographically an increased incorporation of 35Slabelled sulphate into chicken embryo skin under the influence of vitamin A. Lawrence and Ricketts [9] had previously studied the uptake of 35S sulphate by guinea pig skin respiring in vitro, and shown that it could be influenced by heating, bacterial toxins, cortisone or iodoacetic acid. On this basis they concluded that sulphate incorporation was an active enzymatic process and that it was being used for the synthesis of sulphated mucopolysaccharides. Initial studies in this laboratory of organ cultures of adult guinea pig ear skin growing with and without vitamin A, failed to demonstrate any histological effect. Since this might have been due to the fact that the turnover of mucopolysaccharides was likely to be slower in adult than in chicken embryo skin or that the extent to which the adult epidermal cell was capable of undergoing metaplasia was limited, experiments were devised to determine whether any evidence of an effect by vitamin A upon sulphate incorporation could be shown. MATERIALS
AND METHODS
Skin slices were cut free-hand from the dorsum of the guinea pig ear, and floated for 24 hr at 37°C upon the surface of 1 ml of medium containing 0.05 mc Na,%O, added to horse serum 40 per cent and Hanks’ BSS 60 per cent, in a gas phase of 95 per cent oxygen and 5 per cent carbon dioxide. Thin slicesof human skin obtained from excessmaterial used at grafting operations were treated similarly. On removal from the medium, the skin slices were plunged into liquid nitrogen for 30 min, and then dialyzed against three changes of 0.1 M sodium sulphate over 24 hr to remove any surface adsorbed radioactive sulphate. The skin was then rinsed in distilled water and dried to constant weight. Complete destruction of the skin Experimental
Cell
Research
35
256
S. A. Barker,
C. N. D.
Cruickshank
and
Tessa
Webb
fragments in 1 ml of 12 N hydrochloric acid at 100°C was followed by precipitation of the sulphate by addition of 0.1 M sodium sulphate (4 ml) as carrier, followed by 0.1 M barium chloride (5 ml). The solid barium sulphate wasthen placed as an acetone suspensionon to an aluminium planchette. Counting was by the end window method, at the calculated infinite thickness. In someexperiments the epidermis was separated from the dermis after incubation with 0.25 per cent trypsin for 30 min or longer if necessary,and the two tissuestreated separately as above. Further portions of skin were subjected to an extraction procedure to demonstrate the degree of incorporation of the sulphate into mucopolysaccharide. Weighed whole skin was cut into small pieces and extracted with acetone to give a fat fraction. The ground residue was suspended in 0.02 M acetate buffer pH 6, treated with ficin (IO mg) in the presence of L-cysteine as activator and placed upon a slow shaker at 37°C for 24 hr. The digest obtained was extracted three times with a chloroformamyl alcohol mixture (10 : 1 v/v). Protein I was recovered from the organic phase. Addition of 70 per cent perchloric acid (1 ml) to the aqueous phase (20 ml) at 4°C for 30 min gave a precipitate designated protein II. After dialysis of the supernatant, freeze drying the centrifuged solution gave a crude mucopolysaccharide fraction, as shown by staining with alcian blue [8] after separation by paper chromatography in 0.1 M borate buffer pH 10 : ethanol (65 : 35 v/v) or paper electrophoresis in 0.2 M formate buffer pH 2. Acid hydrolysates of the fraction gave positive tests for uranic acid [4] and hexosamine [15]. The specific activity of each fraction was measured exactly as for whole skin. RESULTS
Table I demonstrates that the majority (over 60 per cent) of the sulphate is incorporated into mucopolysaccharide, and that the presence of vitamin A 10 IU/ml= 10-S M, almost doubles the specific activity of the mucopolyTABLE
I. The incorporation of 35S-sulphate into various skin fractions (counts/g/set X 103). Specificactivity Fraction
Vitamin A
Control
4.5 4.9 0.35 0.34 0.23 0.28 0.53 0.83
5.8 7.8 0.46 0.32 0.24 0.31 0.37 0.36
(1) Fat
Experimental
(2)
Protein I
(3)
Protein II
(4)
Mucopolysaccharide
Cell Research
35
% Distribution of activity 19 8 12 61
Sulphafe
mefabolism
in skin
saccharide. Fat appears to have a high specific activity which is lowered by the presence of the vitamin. The amount of fat present, however, constitutes such a small proportion (N 1 per cent) of the whole, that any surface adsorbed 35SOi- ion would considerably magnify the value obtained as specific activity. Table II shows that there is a significantly increased incorporation of sulphate into whole skin slices of guinea pig ear in the presence of vitamin A. It also demonstrates that citral 10-E M has no effect upon the mucopolysaccharides of whole skin and inhibits the vitamin A effect. when the epidermis A considerably different picture emerges, however, and dermis are considered separately. Tables III and IV shorn that for guinea pig and human skin respectively the vitamin A effect is confined entirely to the epidermis and that its presence makes no difyerence to the sulphate TABLE
II.
The effects of vitamin A and citral on whole guinea pig ear (countslglsec X 103).
Experiment No.
A
Control
Citral
Vitamin A and citral
1 2
7.33 f 0.76 5.28 I? 0.82
4.36 i 0.20 3.69k0.16
-
-
3 4
5.94 Yc 0.87 5.20 k 0.10
3.40 F 0.41 2.72 zk 0.09
4.09 k 0.46 2.31 f 0.63
4.12 + 0.12 2.25 IL 0.15
Each
TABLE
Vitamin
skin slices of
III.
value
is mean
f S.E. of three
The effects of vitamin A and citral on guinea and dermis (countslglsec x 103).
Experiment
No.
Vitamin
1 Epidermis Dermis 2 Epidermis Dermis 3 Epidermis Dermis 4 Epidermis Dermis Mean
observations.
of paired
0.78 5.9 1.32 2.5 1.07 3.2 2.1 7.4
A
Control
Citral
0.31 5.5 0.68 2.8
-
0.80 3.6 1.3 5.3
0.66 4.1 1.2 8.3
pig
epidermis
Vitamin A and citral 0.96 3.3 2.3 6.9
observations. Experimental
Cell
Research
35
S. A. Barker,
258
C. N. D. Cruickshank
and Tessa Webb
incorporation of the dermis. Citral, on the other hand, causes a slight increase of sulphate incorporation into dermal polysaccharides, but has no effect on that in the epidermis, which is increased as usual in the presence of vitamin A. Table V shows that hydrocortisone 10-S M has two separate effects, the first to decrease sulphate incorporation into the dermis and the second to act as a IV.
TABLE
The efects of vitamin A and citral on human epidermis and dermis (countslglsec x 103).
Experiment
No.
Vitamin
1 Epidermis Dermis 2 Epidermis Dermis 3 Epidermis Dermis Mean
A
Control
0.22 0.21 4.4 2.7 10.4 1.6
of paired
Citral
Vitamin A and citral
0.14 0.19
-
-
3.0 2.9 8.1 1.6
2.3 3.8 7.9 2.1
2.3 3.8 9.4 1.5
observations.
direct inhibitor of the vitamin A effect upon the epidermis. That these alterations are specific is indicated in Table VI by the absence of any effect by stilboestrol. DISCUSSION
The results obtained in the present study indicate that although no histological changes are detectable in adult skin treated in vitro for a short period V. The effects of vitamin A and hydrocortisone on human epidermis and dermis (counts/g/set X 103).
TABLE
Experiment No. 1 Epidermis Dermis 2 Epidermis Dermis 3 Epidermis Dermis Mean Experimental
Vitamin
Control
Hydrocortisone
9.3 2 0.2 1.4 + 0.4 16.1+ 2.8 6.0 5 0.2 10.4iO.l l.SkO.4
7.8 i: 0.3 1.4kO.2 11.5F0.8 4.8 k 0.5 S.Oi;O.5 1.6k0.2
8.2 * 0.4 0.6kO.l 11.3 k 2.9 2.4 + 0.5 7.8 f 0.6 1.0+0.1
+ S.E. of three Cell Research
observations. 35
Vitamin A + hydrocortisone 7.7 k 0.5 0.6 + 0.0 11.4i:o.5 2.7kO.l 7.2 i. 0.4 1.1 io.1
Sulphate
metabolism
in skin
with excess vitamin A, considerable changes in caused. The increased incorporation of 35S sulphate makes it likely that more polysaccharide is actually evidence for this is at present insufficient. It is of the effect of vitamin A is confined to the epidermis, TABLE
Experiment
Guinea
pig
Human
(1)
Human
(2)
Each
value
VI.
259
the metabolism may be into mucopolysaccharide being produced but the considerable interest that although the same muco-
The effects of vitamin A and stilboestrol on human and guinea pig skin (countslglsec X 103). No.
Vitamin 7.0 k 0.7 9.3kO.l 1.4 IL 0.4 29.5 I! 2.4 4.5 IO.2
(1)
Epidermis Dermis Epidermis Dermis is mean
A
&SE.
of three
Control 3.1+ 0.54 7.8 2 0.7 1.4f0.2 22.6 k 0.6 4.8 i 0.8
Stilboestrol
Vitamin A+ stilboestrol
3.1+ 0.3 s.1* 0.4 1.1 kO.01 19.Ok2.0 5.4 k 0.4
6.6 I!I 0.47 8.5 * 0.1 1.2kO.l 23.5 f 0.6 5.8 t 0.1
observations.
polysaccharides appear to be present in both tissues. This implies that its action is mediated by the epidermal cells and is not directly upon the enzymes involved in sulphation. The apparently double effect of hydrocortisone would appear also to be of some importance, for although it inhibits the increased sulphate uptake caused by vitamin A in the epidermis, it independently lowers the uptake of sulphate in the dermis but not the epidermis of non-vitamin A treated skin. The ability of citral to have an independent effect, namely to increase the uptake of dermal sulphate would seem worthy of further investigation. Citral, a terpcne, has a similar structure to that of part of the vitamin A molecule, and the question arises as to whether further allied compounds might also influence sulphate uptake. There are no comparable studies of sulphate metabolism in relation to the polysaccharides of skin, but the effects of vitamin A upon 35SOi- metabolism in rat colon have been studied. Wolf and Varandani [16] have shown that the lowered incorporation in vitro of inorganic 35S sulphate into colon segments obtained from vitamin A deficient rats can be compensated for by the addition of vitamin A. Moretti and Wolf [ 111have shown vitamin A deficiency in rats causes a lowering of the amount of bound hexosamine in colon segments. Later work on pig colon mucosa [171 implicated a protein fraction precipiExperimental
Cell Research
35
260
S. A. Barker,
C. N. D. Cruickshank
and Tessa Webb
tating at pH 5.2 in this effect on mucopolysaccharide metabolism and the fraction was used to demonstrate 353’-phosphoadenosine-5’-phosphosulphate production, and transfer of 35S sulphate to p-nitrophenol. These workers also found that pre-incubation of the pH 5 enzyme fraction with lipoxidase lowered the overall mucopolysaccharide synthesis, but did not affect the transfer mechanism, indicating that vitamin A has an effect upon the initial activation system of 35S sulphate, as follows: ATP+S Adenosine-5’-phosphosulphate + ATP 4 3’-phosphoadenosine-5’-phosphosulphate
+ pyrophosphate
+ ADP
Vitamin A also has an effect upon the liberation of hydrolytic enzymes contained in a latent state within the lysosome particles of tissues. Such particles generally contain proteases, enzymes able to degrade mucopolysaccharides such as sulphatases, glucosaminidases and glucuronidases as well as nucleases and acid phosphatases [2, 31. Generally, all such enzymes are spilled out almost simultaneously by reagents such as digitonin, detergents and freezing and thawing. Furthermore, vitamin A has also been found to cause the liberation of a protease from the lysosomes of chick cartilage [5]. It is this effect of vitamin A upon the lysosomes which is believed to be inhibited by cortisone and hydrocortisone [a]. This is supported by the work of Fell and Thomas [7] who found that hydrocortisone inhibited the in uitro dissolution of cartilaginous long bone rudiments of chick embryo brought about by organ culture with excess of vitamin A. Apart from this, hydrocortisone causes structural alterations in mast cells [ 11, while its local application on to experimental wounds in rabbit skin, in some cases diminished ground substance metachromasia, degranulated the mast cells and reduced their number [18]. Other workers [IO] have shown that a concentration of hydrocortisone in excess of 0.1 mg/ml of medium suppresses the fixation of labelled sulphate by heart and skeletal muscle. The same workers, however, found that similar concentrations of the steroid had no effect upon 35S sulphate incorporation by liver, and expressed the belief that this indicated a different mechanism. Little is known of the metabolic function of citral, save that it has been found not to be a vitamin A inhibitor in cases of glaucoma [14]. At first glance this does not appear to be true of skin, but closer inspection reveals that while Experimental
Cell Research
35
Sulphate
metabolism
in skin
the vitamin A effect lay in the epidermis, the effect of citral was apparently independent as it lay in the dermis only. It remains to be discovered whether there is any relationship between the two. Although oestradiol has been reported as inhibiting 35S sulphate incorporation into tissue [13], we found stilboestrol to have no effect. SUMMARY
I. Over 60 per cent of the 35S sulphate taken up by skin was shown to be contained in a mucopolysaccharide fraction. 2. Vitamin A caused the specific activity of the mucopolysaccharide fraction to be nearly doubled. 35S sulphate incorporation lay 3. The ability of vitamin A to increase entirely in the epidermis. 1. Hydrocortisone, while inhibiting the vitamin A effect in the epidermis, also caused the 35S sulphate uptake in the dermis to be lowered. We are grateful for the invaluable technical assistance of Miss Gabriel West-Samuel. One of us (T.W.) is in receipt of a University of Birmingham Scholarship. REFERENCES 1. ASBOE HANSEN, C., PhysioZ. Reu. 38, 446 (1958). 2. DE DUVE, C., Biochem. Pharmacol. 9, 97 (1962). 3. DINGLE, J. T., Biochem. J. 79, 509 (1961). 4. DISCHE, Z., J. Mol. Chem. 167, 189 (1947). 5. FELL, H. B. and DIPI’GLE, J. T., Biochem. J. 87, 403 (1963). 6. FELL, H. B. and MELANBY, E., J. Physiol. 119, 470 (1953). 7. FELL, H. B. and THOMAS, J., J. ExptZ Med. 114, 343 (1961). 8. HEREMANS, J. and VAERMAN, J. P., Clin. Chim. Acta 3, 430 (1958). 9. LAWRENCE, J. C. and RICKETTS, C. R., Exptl Cell Res. 12, 633 (1957). 10. LAYTON, L., Proc. Sot. Exptl BioZ. Med. 76, 596 (1951). 11. MORETTI, A. and WOLF, G., Biochim. Biophys. Acla 53, 263 (1961). 12. PELC, S. R. and FELL, H. B., ExptZ Cell Res. 19, 99 (1960). 13. PRIEST, R. E. and KOPLITZ, R. M., J. ExptZ Med. 116, 565 (1962). 14. REDGER, F., SCUDUZZAFAR, M. and DUYAL, Y., Trans. Ophthal. Sot. 79, 283 (1959). 15. RONDLE, C. and MORGAN, M., Biochem. J. 61, 586 (1955). 16. WOLF, G. and VARANDANI, P., Biochim. Biophys. Acta 43, 501 (1960). 17. WOLF, G., VARANDANI, P. and JOHNSON, B. C., Riochim. Biophys. Acta, 46, 59 (1961). 18. ZACHARIAE, L. and MOLTKE, E., Acta Endocrinol. 16, 300 (1954).
Experimental
Cell
Research
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