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The incorporation of phosphate by mammalian skin in tissue culture
587 THE
INCORPORATION
OF PHOSPHATE IN TISSUE
SHIRLEY Medical
A. CARNEY, Research Birmingham
BY MAMMALIAN
CULTURE
J. C. LAWRENCEand
C. R.
RICKETTS
Council Industrial Injuries & Burns Research Accident Hospital, Birmingham, England Received
SKIN
April
Unit,
17, 1962
THE ability of skin to take up phosphate ions from a culture medium in vitro has been used by Gemmell, Laing and Veal1 [3] as the basisof a method of assessingviability. Lawrence and Ricketts [5] have shown that in mammalian skin damaged by heat, respiration and uptake of sulphate ions are diminished. It was therefore of interest to extend this study by investigating the effect of thermal damage on the incorporation of phosphate into cell constituents. Methods.-Slices of skin weighing 5-7 mg were floated on sterile physiological saline [S] and maintained at selected temperatures for 30 min periods. The sliceswere then transferred to the medium of homologous serum, glucose and buffer solution as described by Lawrence [4] but containing 2 &/ml of S2P-orthophosphate, and incubated at 37°C for 24 hr. For measurementof S2P-phosphateuptake the slicesof skin were washedthree times with distilled water and dissolved in 60 per cent perchloric acid. In somecasestotal phosphorus was determined at this stage by the method of Allen [I]. The solutions were then made up to known volume and the radioactivity was measuredin a Geiger counter for liquid samples. In experiments to measure the incorporation of S2P-phosphateinto various constituents, the skin sampleswere fractionated by the method of Schneider [6] and the radioactivity of the individual fractions was measured. All measurements were related to the fresh weight. I. The effect of incubation
TABLE
lime on the incorporaiion guinea-pig skin.
of 3zP-phosphafe
info
Standard
Meanphos- Standard Mean phorus error of mean Incubation Kumberof Meancounts/ of mean radioactivity content in phosphorus in PCx lo5 time in hr experiments min/mg counts content /%/mg tTr0r
2
655
+13
29.51
2.60
kO.15
4
1061
+25
47.79
2.22
20.12
G
1231
-t18
55.45
2.09
+0.10
8
1588
kll8
71.53
2.29
kO.33
12
18Gl
f36
83.83
2.22
i0.26
16
2060
ilO
92.79
20
2420
2146
24
1889
2342
109.00 85.09
not determined 1.98 -i-o.21 1.37 io.14 Experimental
Cell Research
27
Shirley A. Carney, J. C. Lawrence and C. R. Rickelfs SzP-phosphate content of guinea-pig skin incubated at 35°C at The increase in specific radioactivity reached a steady rate during the latter part of the incubation period (Table I, Fig. l), and the total phosphorus content remained at about 0.21 per cent. Experiments in which 24hr skin cultures were heated at 60°C for 10 min to inactivate enzymes and then dialysed Results.--The first increased
rapidly.
Fig. l.-The effect of incubation time on the specific radioactivity of phosphorus in guinea-pig skin at 37’C. Specific radioactivity is obtained by dividing radioactivity by phosphorus content, see Table I.
8 10 12 16 1 iocubotlo” tmeI” hours
against isotonic phosphate buffer, pH 7, showed that only 25 per cent of the total phosphorus was removed by dialysis, but that this fraction contained 79 per cent of the radioactivity, indicating material of high specific activity. Table II and Fig. 2 record incorporation of S2P-phosphateinto skin after heat pretreatment at various temperatures. Inhibition values were based on the sample maintained at 37°C (0 y0 inhibition). The curves obtained were sigmoid in form. The steepest portion TABLE
of the curve for guinea-pig
II. The effect of thermal Guinea-pig
damage
on S2P-phosphate
-7
r-
skin
Pretreatment temperature, “C
Number of experiments
Mean counts/ min/mg
37
4
3506
40
4
3451
42
4
3224
f 209 59 -t 369
43
4
2916
+244
skin was almost
Standard error of mean counts
parallel
with
the curves
incorporation
by skin.
Human skin Mean counts/ min/mg
Standard error of mean counts
Percentage inhibition
Number of experiments
Percentage inhibition
0 1.8
3
1281
i206
0
4
1171
+144
8.6
8.2
4
25.1
4
960 958
? 93
17.0
i62
25.2
38.8
45
4
1211
IL7
65.5
4
784
251
47
3
632
+363
71.5a
4
544
?:a3
57.5
50
3 3
407
+-92
81.7=
4
470
+46
63.3
37
2217
278
Oa
a Values for guinea-pig skin at 47 and 5O’C wrre obtained in a separate experiment included a control at 37°C. Experimental
Cell
Research.
27
which
Phosphafe
incorporation
by skin
589
obtained by Lawrence and Ricketts [5] for respiration and sulphate uptake inhibition, but occurred at temperatures approximately 1°C higher. The curve obtained for human skin was flatter, reaching only 63 per cent inhibition at 50°C. This may be due to the fact that human skin is less active metabolically than guinea-pig skin [5] and contains more dermis; thus there is a higher proportion of inert material which phosphate ions enter only by diffusion. Values obtained in the experiments so far described have necessarily included radioactivity due to inorganic phosphate. It was not possible to remove this by dialysis since skin is known to contain a large number of water-soluble phosphate esters
Fig. 2.-The effect phate incorporation human skin ( 0 ).
TABLE
Pretreatment tcmperature “C
of thermal damage on =P-phosby guinea-pig skin (0) and
The effect of thermal damage on 32P-phosphate stituents of guinea-pig skin.
III.
Number of experiments
Mean counts/ min/mg l’.C.A.-soluble
37 40 42 44 47 50
3 3 3 3 3 3
720 510 653 262 189 154 Sucleic
37 40 42 44 47 50
5s 58 52 12 1.2 0.8
Standard error of mean counts
Perrentage inhibition
compounds ?I16 ilO i 60 IL 45 I! 26 L!I 13
Mean counts/ min/mg
into cell con-
Standard error of mean counts
Percentage inhibition
Phospholipids 0 1.4 9.3 63.6 73.8 78.6
acids i k i k k k
Number of experiments
incorporation
152 1 .il 148 100 9 3
-21 I21 Xl8 i34 i 1.2 i 0.5
0 0.7 2.6 34.2 94.1 98.0
+3 z!z3 +4 ?6 2 0.6
0 0 14.6 26.8 95.1 100.0
Phosphoproteins 11 11 12 3 0.2 0.1
0 0 10.3 79.3 98.3 98.3
41 41 35 30 2 0 Experimental
Cell Research
27
Shirley A. Carney, J. C. Lawrence and C. R. Ricketts [2] which would also be lost. Guinea-pig skin was therefore fractionated 161 into trichloracetic acid-soluble substances, phospholipids, nucleic acids and phosphoproteins, and the radioactivity of each fraction was measured. Results are shown in Table III and Fig. 3. Parallel sigmoid curves were obtained showing 50 per cent inhibition at differing temperatures, and in three cases reaching almost complete inhibition at 50°C. The T.C.A.-soluble fraction had reached only 79 per cent inhibition at 50°C. Radioactive 0-phosphorylserine was identified in hydrolysates of the phosphoprotein fraction by co-chromatography with an authentic sample in two dimensions on paper using phenol, 0.880 NH,OH, H,O, 160 g:i ml:20 ml and BuOH, HAc, H,O, 120:30:50 by vol.
Fig. S.--The
effect of thermal damage on 32P-
Discussion.-The phosphate uptake of skin in tissue culture has been shown to exhibit a graded response to thermal damage over the range 37-50°C. The sigmoid uptake probably indicates a normal form of the graphs of total 32P-phosphate distribution of the heat sensitivities of individual cells. This hypothesis is consistent with previous experiments [5] on the inhibition of sulphate uptake and respiration. The graphs showing incorporation into different cell fractions are also sigmoid but do not coincide. This suggests that in addition to the variations exhibited by individual cells as units, the enzyme systems concerned in the synthesis of phosphorus compounds also differ in their stability to heat. In this connection it is interesting to note that the systems synthesising nucleic acids and soluble esters (chiefly sugar phosphates and nucleotides [a]) are apparently more heat labile than those incorporating s2P-phosphate into phospholipids and phosphoproteins. The curves for total P uptake, Fig. 2, do not approach 100 per cent inhibition at 50°C. Considering the curves for the fractions which form the total, Fig. 3, it is clear that the anomaly is due to the T.C.A.-soluble fraction which includes inorganic phosphate. Radioautographs of T.C.A.-soluble compounds from skin, prepared as described by Brooks, Lawrence and Ricketts [2] showed labelled phosphate esters when the damaging temperature maintained for 30 min was 45°C or less. Above 45°C a large amount of inorganic phosphate only was present. Increased permeability of the cell may enable phosphate uptake to continue by diffusion after the cell has suffered thermal damage sufficient to inhibit metabolic uptake. Summary.-Small pieces of skin were exposed to thermal damage for 30 min at temperatures between 37 and 50°C and then maintained on a suitable medium Experimental
Cell Research
27
Phosphate
incorporation
by skin
591
at 37°C. The total uptake of phosphate ions showed graded inhibition, suggesting that the individual cells varied in sensitivity to thermal damage. The inhibition of phosphate uptake into cellular constituents differed at each temperature, suggesting that the enzyme systems differed in their stability to heat. The authors thank Dr. G. B. Ansel for the sample of 0-phosphorylserine. REFERENCES 1. 2. 3. 4. 5. 6.
ALLEN, R. J. L., Biochem. J. 34, 858 (1940). BROOKS. S. A., LAWRENCE. J. C. and RICKETTS, C. R., Biochem. J.73.566 (1959). GEM&L, W.; LAING, J. l?. and VEALL, N., Radioisotope Conference 1, lh7 (1954). LAWRENCE, J. C., Brit. J. Pharmacol. 14, 168 (1959). LAWRENCE, J. C. and RICKETTS, C. R., Exptl. Cell Research 12, 633 (1957). SCHNEIDER, W. C., J. biol. Chem. 161, 293 (1945).
THE
MICROMORPHOLOGY
OF AMOEBA
PRESSURE-INDUCED
CHANGES
SOL-GEL J. V. LANDAU General
Medical
Research
PROTEUS
DURING
IN THE
CYCLE’
and
L. THIBODEAU3
Laboratory, VA Hospital; and Departments of Medicine Albany Medical College, Albany, N.Y., U.S.A. Received
April
and Pathology,
24, 1962
1-r has previously been shown [l] that high hydrostatic pressure has the ability to reversibly disrupt the structural integrity of the plasmagel component of Amoeba proteus. The disruption of the gel results in a loss of pseudopodial activity and the eventual attainment of a spherical shape (Fig. 1 a). Upon release of pressure the cytoplasm undergoes a re-gelation, and at 15 set post-release, an overall contraction (Fig. I b). This contracted state is maintained for 15 set after which membrane activity and pseudopodial flow commence. It was considered of interest to compare the micromorphology of the amoeba during each of the above phases of the sol-gel cycle. This is a report of the preliminary results of such a study. The first problem was to devise a pressure vessel which would permit fixation of the amoeba while under pressure and at any given time interval following the release of pressure. The design of such a vessel is shown in Fig. 2. The entire apparatus 1 Partially 2 Address: 3 Present
supported by U.S.P.H.S. grants, Cy-2664 and C-5054. General Medical Research Laboratory, VA Hospital, address: University of California, Berkeley, California,
Albany, U.S.A. Experimental
Kew
York,
U.S.A.
Cell Research
27
INCORPORATION
OF PHOSPHATE IN TISSUE
SHIRLEY Medical
A. CARNEY, Research Birmingham
BY MAMMALIAN
CULTURE
J. C. LAWRENCEand
C. R.
RICKETTS
Council Industrial Injuries & Burns Research Accident Hospital, Birmingham, England Received
SKIN
April
Unit,
17, 1962
THE ability of skin to take up phosphate ions from a culture medium in vitro has been used by Gemmell, Laing and Veal1 [3] as the basisof a method of assessingviability. Lawrence and Ricketts [5] have shown that in mammalian skin damaged by heat, respiration and uptake of sulphate ions are diminished. It was therefore of interest to extend this study by investigating the effect of thermal damage on the incorporation of phosphate into cell constituents. Methods.-Slices of skin weighing 5-7 mg were floated on sterile physiological saline [S] and maintained at selected temperatures for 30 min periods. The sliceswere then transferred to the medium of homologous serum, glucose and buffer solution as described by Lawrence [4] but containing 2 &/ml of S2P-orthophosphate, and incubated at 37°C for 24 hr. For measurementof S2P-phosphateuptake the slicesof skin were washedthree times with distilled water and dissolved in 60 per cent perchloric acid. In somecasestotal phosphorus was determined at this stage by the method of Allen [I]. The solutions were then made up to known volume and the radioactivity was measuredin a Geiger counter for liquid samples. In experiments to measure the incorporation of S2P-phosphateinto various constituents, the skin sampleswere fractionated by the method of Schneider [6] and the radioactivity of the individual fractions was measured. All measurements were related to the fresh weight. I. The effect of incubation
TABLE
lime on the incorporaiion guinea-pig skin.
of 3zP-phosphafe
info
Standard
Meanphos- Standard Mean phorus error of mean Incubation Kumberof Meancounts/ of mean radioactivity content in phosphorus in PCx lo5 time in hr experiments min/mg counts content /%/mg tTr0r
2
655
+13
29.51
2.60
kO.15
4
1061
+25
47.79
2.22
20.12
G
1231
-t18
55.45
2.09
+0.10
8
1588
kll8
71.53
2.29
kO.33
12
18Gl
f36
83.83
2.22
i0.26
16
2060
ilO
92.79
20
2420
2146
24
1889
2342
109.00 85.09
not determined 1.98 -i-o.21 1.37 io.14 Experimental
Cell Research
27
Shirley A. Carney, J. C. Lawrence and C. R. Rickelfs SzP-phosphate content of guinea-pig skin incubated at 35°C at The increase in specific radioactivity reached a steady rate during the latter part of the incubation period (Table I, Fig. l), and the total phosphorus content remained at about 0.21 per cent. Experiments in which 24hr skin cultures were heated at 60°C for 10 min to inactivate enzymes and then dialysed Results.--The first increased
rapidly.
Fig. l.-The effect of incubation time on the specific radioactivity of phosphorus in guinea-pig skin at 37’C. Specific radioactivity is obtained by dividing radioactivity by phosphorus content, see Table I.
8 10 12 16 1 iocubotlo” tmeI” hours
against isotonic phosphate buffer, pH 7, showed that only 25 per cent of the total phosphorus was removed by dialysis, but that this fraction contained 79 per cent of the radioactivity, indicating material of high specific activity. Table II and Fig. 2 record incorporation of S2P-phosphateinto skin after heat pretreatment at various temperatures. Inhibition values were based on the sample maintained at 37°C (0 y0 inhibition). The curves obtained were sigmoid in form. The steepest portion TABLE
of the curve for guinea-pig
II. The effect of thermal Guinea-pig
damage
on S2P-phosphate
-7
r-
skin
Pretreatment temperature, “C
Number of experiments
Mean counts/ min/mg
37
4
3506
40
4
3451
42
4
3224
f 209 59 -t 369
43
4
2916
+244
skin was almost
Standard error of mean counts
parallel
with
the curves
incorporation
by skin.
Human skin Mean counts/ min/mg
Standard error of mean counts
Percentage inhibition
Number of experiments
Percentage inhibition
0 1.8
3
1281
i206
0
4
1171
+144
8.6
8.2
4
25.1
4
960 958
? 93
17.0
i62
25.2
38.8
45
4
1211
IL7
65.5
4
784
251
47
3
632
+363
71.5a
4
544
?:a3
57.5
50
3 3
407
+-92
81.7=
4
470
+46
63.3
37
2217
278
Oa
a Values for guinea-pig skin at 47 and 5O’C wrre obtained in a separate experiment included a control at 37°C. Experimental
Cell
Research.
27
which
Phosphafe
incorporation
by skin
589
obtained by Lawrence and Ricketts [5] for respiration and sulphate uptake inhibition, but occurred at temperatures approximately 1°C higher. The curve obtained for human skin was flatter, reaching only 63 per cent inhibition at 50°C. This may be due to the fact that human skin is less active metabolically than guinea-pig skin [5] and contains more dermis; thus there is a higher proportion of inert material which phosphate ions enter only by diffusion. Values obtained in the experiments so far described have necessarily included radioactivity due to inorganic phosphate. It was not possible to remove this by dialysis since skin is known to contain a large number of water-soluble phosphate esters
Fig. 2.-The effect phate incorporation human skin ( 0 ).
TABLE
Pretreatment tcmperature “C
of thermal damage on =P-phosby guinea-pig skin (0) and
The effect of thermal damage on 32P-phosphate stituents of guinea-pig skin.
III.
Number of experiments
Mean counts/ min/mg l’.C.A.-soluble
37 40 42 44 47 50
3 3 3 3 3 3
720 510 653 262 189 154 Sucleic
37 40 42 44 47 50
5s 58 52 12 1.2 0.8
Standard error of mean counts
Perrentage inhibition
compounds ?I16 ilO i 60 IL 45 I! 26 L!I 13
Mean counts/ min/mg
into cell con-
Standard error of mean counts
Percentage inhibition
Phospholipids 0 1.4 9.3 63.6 73.8 78.6
acids i k i k k k
Number of experiments
incorporation
152 1 .il 148 100 9 3
-21 I21 Xl8 i34 i 1.2 i 0.5
0 0.7 2.6 34.2 94.1 98.0
+3 z!z3 +4 ?6 2 0.6
0 0 14.6 26.8 95.1 100.0
Phosphoproteins 11 11 12 3 0.2 0.1
0 0 10.3 79.3 98.3 98.3
41 41 35 30 2 0 Experimental
Cell Research
27
Shirley A. Carney, J. C. Lawrence and C. R. Ricketts [2] which would also be lost. Guinea-pig skin was therefore fractionated 161 into trichloracetic acid-soluble substances, phospholipids, nucleic acids and phosphoproteins, and the radioactivity of each fraction was measured. Results are shown in Table III and Fig. 3. Parallel sigmoid curves were obtained showing 50 per cent inhibition at differing temperatures, and in three cases reaching almost complete inhibition at 50°C. The T.C.A.-soluble fraction had reached only 79 per cent inhibition at 50°C. Radioactive 0-phosphorylserine was identified in hydrolysates of the phosphoprotein fraction by co-chromatography with an authentic sample in two dimensions on paper using phenol, 0.880 NH,OH, H,O, 160 g:i ml:20 ml and BuOH, HAc, H,O, 120:30:50 by vol.
Fig. S.--The
effect of thermal damage on 32P-
Discussion.-The phosphate uptake of skin in tissue culture has been shown to exhibit a graded response to thermal damage over the range 37-50°C. The sigmoid uptake probably indicates a normal form of the graphs of total 32P-phosphate distribution of the heat sensitivities of individual cells. This hypothesis is consistent with previous experiments [5] on the inhibition of sulphate uptake and respiration. The graphs showing incorporation into different cell fractions are also sigmoid but do not coincide. This suggests that in addition to the variations exhibited by individual cells as units, the enzyme systems concerned in the synthesis of phosphorus compounds also differ in their stability to heat. In this connection it is interesting to note that the systems synthesising nucleic acids and soluble esters (chiefly sugar phosphates and nucleotides [a]) are apparently more heat labile than those incorporating s2P-phosphate into phospholipids and phosphoproteins. The curves for total P uptake, Fig. 2, do not approach 100 per cent inhibition at 50°C. Considering the curves for the fractions which form the total, Fig. 3, it is clear that the anomaly is due to the T.C.A.-soluble fraction which includes inorganic phosphate. Radioautographs of T.C.A.-soluble compounds from skin, prepared as described by Brooks, Lawrence and Ricketts [2] showed labelled phosphate esters when the damaging temperature maintained for 30 min was 45°C or less. Above 45°C a large amount of inorganic phosphate only was present. Increased permeability of the cell may enable phosphate uptake to continue by diffusion after the cell has suffered thermal damage sufficient to inhibit metabolic uptake. Summary.-Small pieces of skin were exposed to thermal damage for 30 min at temperatures between 37 and 50°C and then maintained on a suitable medium Experimental
Cell Research
27
Phosphate
incorporation
by skin
591
at 37°C. The total uptake of phosphate ions showed graded inhibition, suggesting that the individual cells varied in sensitivity to thermal damage. The inhibition of phosphate uptake into cellular constituents differed at each temperature, suggesting that the enzyme systems differed in their stability to heat. The authors thank Dr. G. B. Ansel for the sample of 0-phosphorylserine. REFERENCES 1. 2. 3. 4. 5. 6.
ALLEN, R. J. L., Biochem. J. 34, 858 (1940). BROOKS. S. A., LAWRENCE. J. C. and RICKETTS, C. R., Biochem. J.73.566 (1959). GEM&L, W.; LAING, J. l?. and VEALL, N., Radioisotope Conference 1, lh7 (1954). LAWRENCE, J. C., Brit. J. Pharmacol. 14, 168 (1959). LAWRENCE, J. C. and RICKETTS, C. R., Exptl. Cell Research 12, 633 (1957). SCHNEIDER, W. C., J. biol. Chem. 161, 293 (1945).
THE
MICROMORPHOLOGY
OF AMOEBA
PRESSURE-INDUCED
CHANGES
SOL-GEL J. V. LANDAU General
Medical
Research
PROTEUS
DURING
IN THE
CYCLE’
and
L. THIBODEAU3
Laboratory, VA Hospital; and Departments of Medicine Albany Medical College, Albany, N.Y., U.S.A. Received
April
and Pathology,
24, 1962
1-r has previously been shown [l] that high hydrostatic pressure has the ability to reversibly disrupt the structural integrity of the plasmagel component of Amoeba proteus. The disruption of the gel results in a loss of pseudopodial activity and the eventual attainment of a spherical shape (Fig. 1 a). Upon release of pressure the cytoplasm undergoes a re-gelation, and at 15 set post-release, an overall contraction (Fig. I b). This contracted state is maintained for 15 set after which membrane activity and pseudopodial flow commence. It was considered of interest to compare the micromorphology of the amoeba during each of the above phases of the sol-gel cycle. This is a report of the preliminary results of such a study. The first problem was to devise a pressure vessel which would permit fixation of the amoeba while under pressure and at any given time interval following the release of pressure. The design of such a vessel is shown in Fig. 2. The entire apparatus 1 Partially 2 Address: 3 Present
supported by U.S.P.H.S. grants, Cy-2664 and C-5054. General Medical Research Laboratory, VA Hospital, address: University of California, Berkeley, California,
Albany, U.S.A. Experimental
Kew
York,
U.S.A.
Cell Research
27