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The value of intact tissue sections for studying metabolic inter-actions between the cytoplasm and mitochondria
0
656
Experimental
1968 by Academic
Press Inc.
Cell Research 49, 656-665 (1968)
THE VALUE OF INTACT TISSUE SECTIONS FOR STUDYING METABOLIC INTER-ACTIONS BETWEEN THE CYTOPLASM AND MITOCHONDRIA R. G. BUTCHER Division
of Cellular
Biology,
and J. CHAYEN
The ~a~hilda and Terence Kennedy London W6, UK
Institute
of Rheumatology,
Received May 26, 1967l
The metabolic activities of mitochondria and of the other parts of the cytoplasm are most commonly studied by homogenizing the cells and separating the various fractions. It is well understood that these procedures may alter the activities of the parts so isolated. Moreover it is obvious that, after such procedures, any interactions between the parts can be calculated only by inference from the activities of the separate parts. This type of inference has an inherent assumption, namely that the metabolism of the cell is approximately the sum of the activities of the separate systems. Because this assumption, and these inter-actions, are of such importance, techniques have been devised which retain the normal spatial arrangement of cellular organelles, and the less particulate cytoplasm, even during biochemical study. Tissue is super-cooled [13] and sectioned at S-20 /J under such conditions that this super-cooled state is maintained [ 151. The sections are incubated in relatively conventional biochemical media; but to ensure that the non-denatured protoplasm is not solubilised during such incubation, a sufficient concentration of polyvinyl alcohol (PVA) must be incorporated into the media [1, 2 j. The validity of these procedures has been discussed fully (e.g. [9, 10, 111; also [3, 4] in relation to lysosomal integrity). Thus it is now possible to prepare sections of tissue in which the histological and even the cytological structure [5] is intact and to retain the tissue intact while it is tested for metabolic activity. Dehydrogenase activity can be measured quantitatively in such sections if neotetrazolium is used as the hydrogen-acceptor [14]; the deposit of reduced neotetrazolium (formazan) is eluted and measured spectrophotometrically. Direct proportionality between the amount of dehydrogenation and the amount of formazan so produced has been demonstrated [la] and the results can then be expressed 1 Revised version received September 26, 1967. Experimental
Cell Research 49
Intact
tibiae sections and ~et~~ol~c inter-acfiorl~
657
against unit area, or unit tissue nitrogen, or wit amount of tissue nucleic acid [?, $11. The crucial test of whether such procedures have a signi~cant place in biochemistry is whether they can give direct information concerning interactions between different parts of cells, whereas such ~rlter-actions might be espected to be distorted or to be derivable only by inference by conventional biochemical procedures. Of especial interest has been the apparent need for oxidation associated with the pentose shunt in maintaining the integrity of the more soluble parts of these sections [12]. Therefore it seemed that a rigorous test would be to examine a l~ito~holldrial system, namefy the oxidation of glutamate, and to see if this was affected by cytoplasmic oxidation. Moreover, the results with P\rA meant that any interacting systems could be investigated when in the iutact section (i.e. in the presence of WA) and compared to \vhat happens in the relatively homogenisetf tissue (namely the same type of system but in the absence of P\‘A). MATERIALS
AND
METHODS
Two strains of rats were used: albino Wistar rats and inbred August rats. They were killed by nitrogen and small pieces (about 0.5 cma) of their livers were chilled in hexane at about - 70°C. Sections (B-14 ,u) were cut at an ambient temperature of between - 25” and - 30°C with a chilled knife [ll, 15]; they were partially dried by the fact that they were picked off the knife on to a warm slide [Ill. At laboratory temperature, a ring of Perspex was sealed around each section by means of a film of Yaseline [6, II]. A measured volume of incubation medium, previously equilibrated to 37X, was put into the ring; after a standard time the medium and the ring were removed, the sect.ions were rinsed in distilled water and dried. The area of the section was measured by planimetry of the image, projected through a photographic enlarger. The Pormazan in the section was then eluted into 0.25 ml of 10 per cent nheptanol in sgm-tetrachloroethane 1121 and measured in microcells in a Hilger spectrophotometer at 559 m/J. The amount of formazan was determined by reference to a standard calibration graph and was related to unit area (by planimetry) and thus to unit volume of tissue assuming the thickness of the section to be that shown on the setting of the microtome. (This assumption was made on the basis of previous studies which related ~lominal thickness to amount of tissue nitrogen [7].) For each (determination four sections were measured and the result expressed as the mean value and standard deviation. The latter also constitutes a check on the conformity of thickness. All incubation media contained 0.1 per cent Ileotetrazolium chloride (Universal Crop Protection) in 0.05 M glycyl-glycine buffer at pH 8.0. To some of these was added 20 per cent w/v of polyvinyl alcohol (PVA: polyviol MO5~1~0 from Bush, Beach & Segner Bayley). According to the system to be tested, the incubation medium contained all or various combinations of the following: 0.15 m&ml glucose-6phosphate (disodium salt; Boehringer); 0.25 m&ml N,4DP+ (Roehringer); 5 mg/rnX
658
R. G. Butcher and J. Chayen
(or in specified cases 0.25 mg/ml) NAD+ (Boehringer}; 17 mg/ml L-glutamic acid (sodium salt; Lights). In other media the glucose-6-phosphate and NADP+ were replaced by 0.25 mg/ml NADPH (Boehringer). In some media the glucose-6-phosphate was replaced by 0.15 mg/ml 6-phosphogluconate (trisodium salt; Wessex). Cytochrome oxidase was demonstrated by the method of Butcher et crl. [8] at pH 8.0 and 37°C. In some experiments 20 per cent PVA was added to this medium. RESULTS
Comparison of g~l~tarnate and ~la~ose-6-phosphate studied singly or in combination
dehydrogenation
when
In these experiments the concentration of glucose-6-phosphate and of NADP+ has been kept to l/l0 optimal in an attempt to clarify the mechanism by which this pen&se-shunt system inter-acts with the glutamate dehydrogenase system. Consequently the activity shown by the tissue when incubated in this medium was consistently low (line 2, Tables 1 and 2). In most experiments the amount of oxidation of glutamate was enhanced if tested in the presenee of PVA (compare line 3, Tables 1 and 2). The sum of these two separate deh~drogenase systems (line 2 +line 3) never equalled the activity shown when serial sections from the same block of tissue were incubated in a medium containing the same concentration of both substrates and both co-enzymes (line 1, Tables 1 and 2). The difference between the total activity and the sum of the separate activities (line 4, i.e. lines l-(2 +3) was always greater when tested in the presence of PVA than in its absence (comparison of line 4 in Table 1 against line 4 in Table 2). This is especially apparent when the results from the same animal are compared (line 4, Tables 1 and 2, e.g. Expt Nos 15 and 33). TABLE
1. The actiuities of glucose-6-phosphate and glutamate when studied singly and in combination. Incubations
in the presence
dehydrogenases
of PVA.
pg Formazan/mm3/h Experiment Strain
1. G-6-P
+ NADPf
2. G-6-P
+ iXADP+
3. GLUT
+ NADf
number of rat . . .
+ GLUT
.. .
10 Albino
-t NAT)+
17.5 ci: 0.9 0.6 i: 0.04 3.4io.3
4. l-(2+3) G-6-P
13.5 = glucose-6-phosphate.
Experimental
GLUT
Cell Research 49
= glutamate.
38 Albino
33 August
36 August
3s August
27.3k 1.3 0.6 IO.06 2.0f0.15
2&I&0.6
27.153.9
32.2k1.6
24.0+1.:
1.OiO.l 12.1k1.7
lO.SkO.4
17.2k2.2
0.9 ILo.: 13.030.:
24.7
15.3
16.2
14.6
10.1
15 Albino
0.3 io.04
0.4 rf: 0.01
Intact TABLE
tissue sections and metabolic inter-actions
2. The activities of glucose-6-phosphate and glutamate when studied singly and in combination. Incubations
659 dehydrogenases
in the absence of PVA.
pg Formazan/mm3/h Experiment number . . . Strain of rat . 1. G-6-P + NADP+ GLUT + NAD+
1 Albino
2 Albino
3 Albino
10 Albino
15 Albino
16.9kO.9 1.2kO.l 3.8 io.3 11.9
11.9k0.4 1.7kO.l 3.0&0.1 7.2
13.6kO.7 1.4kO.2 2.2 +o.n 10.0
11.7F0.6 0.6iO.l 2.lkO.2 9.0
33 August
34 August
f
2. G-6-P + NADPf 3. GLIJT + Nr\D+ -1. 1 - (2 + 3)
The possibility
15.5 + 0.9 3.3 AZ0.3 2.4 k 0.2 9.8
of a transhydrogenation
phenomenon
9.3f0.7 9.3kO.6 0.3 + 0.06 0.2 &- 0.02 1.3iO.l l.Ot-0.12 8.0 7.8
in the combined system
IVith two substrates and two co-enzymes there were 14 possible permutations which could be contributing to this combined activity. Many have been tested for some of the tissues (Tables 4 and 5) but all have been tested at the same time on one tissue, namely that taken from animal No. 33. To do this, sections were incubated in the basic medium, either with or without PVA, to which one, two three or all four of the factors (either substrate or co-enzyme) were added and the amount of dehydrogenation produced from the given selection of factors was measured (Table 3). The only unusual combination to produce a significant difference was the addition of NAD+ to the normal NADP+-glucose-6-phosphate medium (compare line 3 with line 2, Table 3). This enhancement of dehydrogenation over that produced from either cofactor or from glucose-6-phosphate with either cofactor, could have two possible explanations. One would be that glucose-B-phosphate dehydrogenase requires both co-enzymes for full activity in such sections; the other would be that there is inter-action between NAD+ and the NADPH generated by the action of glucose-B-phosphate dehydrogenase. To test this, NADPH was substituted for glucose-6-phosphate and NL4DP+, in the presence of NAD+. The presence of NADPH together with NAD+ produced considerably more formazan than did either tested alone (comparison of line 7 and line 8 with line 5 in Table 4; also line 7 and line 9 with line 6 in Tables 4 and 5). In another experiment 6-phosphogluconate was substituted for glucose-6phosphate, at equimolar concentration, and this too showed the enhanced effect of having both co-enzymes present, so indicating that it was predominantly an inter-action of NPlDPH with NAD+, irrespective of how the Experimental
Celt Research 49
N. G. Butcher
660 TABLE
and J. Chayen
3. The effect of the various combinations of substrates and coenzymes on disrupted (no PVA) and intact (with 20 y0 PVA) tissue sections ,ug Formazan/mm3/h No PVA 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 23. 14. 15.
G-6-P + NADP’ t GLUT f NAD+ G-G-P I- NADPf G-6-P + NADPI’ i NAD+ GLUT + NAD’GLUT + SAD+, -t- NADP” G-6-P -6 GLI:T i- NAD’ G-6-P + GLIJT i NADP+ G-6-P+ NADf GLI’T/ i- NADP” XADP-‘iSAD-’ G-6-P + GLUT G-6-P SADP’ GLUT NAD+
9.3 *o.i 0.3 iO.06 2.0+0.14
1.0+0.12 1.1 io.03 l.Z&O.Z 1.1+-0.01 0.3 +0.03 0 0.3 & 0.01 0 0 0 0 0.3 20.03
lndophenol
Reduced C,Aachromca,
Cytochrame c
Oxidose (0)
Fig. 1. Eqerimenfal
Cell Research 49
r+
Ox,d,ied Cytochrome
c ’
%’
20 % PTA 27.1 0.3 3.7 10.6 9.3 11.2 1.6 0.5 0 0.5 0 0 0 0 0.5
k3.9 +0.04 kO.4 io.4 10.3 10.3 lo.1 *o.os * 0.05
+0.05
Intact
tissue and sections ll~efaboli~ infer-~~fio~s
661
former was supplied to the system (comparison of lines 4 and 9 with line 3, Tables 4 and 5). The extent of the “transhydrogenation” in the presence and absence of PVA, is given in line 10 in Tables 4 and 5; only in the former, i.e. in the intact tissue, is this el’fert appreciable.
If these results indicate the existence of a true transhydrogenation mechanism then it would be valuable to have a way of testing whether the hydrogen, derived from the pentose-shunt, can be shown inside the mitochondria. An attempt to do this has been made by examining the cytochemical cytochrome oxidase activity shown by these sections. This activity depends on the presence of oxidised cytochrome c: Fig. 1. It therefore seemed reasonable to test whether mitochondrial dehgdrogenase activity could reduce the cytochrome c sufficiently to abolish this cytochemical reaction. To investigate this, serial sections were incubated (,a) in the medium for demonstrating cytochrome oxidase activity alone, or (b) in the same medium to \\-hich enough succinate (0.05 M) was added as would be required for the histochemical demonstration of surcinate dehydrogenase. It was found that the presence of this con~entratio~l of succinate completely inhibite~l the histothemiral demonstration of cytochrome oxidase. In marked contrast, the addition of glucose-g-phosphate and KADP+ to the normal incubation medium for cytochrome oxidase, whether this contained PVA or not, had no effect on the production of the indophenol. Since these results indicated that intra-mitochondrial hydrogen-transport could inhibit the histochemical demonstration of cytoehrome oxidase activity, assurnabiy h;v ll~aintaiiling the ~~t(~chrome c in the reduced state, it seemed reasonable to test the apparent transhy[lrogenati(~ll process to see \\hether it contributed to this mitochondrial pathway. Consequentiy tests were made, in the presence oC 20 per vent PVA, of the elects of adding 0.25 mg/ml NADPH (3 x 1W4 N) and 0.25 mg/ml NAD+ (3.8 x 10V4 M), either singly or together, to the incubation medium for cytochrome oxidase. Each separately had no effect whereas when they were added together practically no stain for cytothrome oxidase was obtained, that is, there was no l~ro(l~l~tion of indophenol. This effect was found only in the presence of PVA; when glucose-6-phosphate and both NADP+ and KAD+ were added to the incubation medimn for cptochrome oxidase but without the addition of PVA, these additional ingredients had no efTect on the normal production of the indophenol. Exi~erirn~rli~l
Ceil Research 49
R. G. Butcher and J. Chayen
662
pg Formazan/mm3/h Experiment number . . . Strain of rat ,.. 1. 2. 3. 4. 5. 6. 7.
G-6-P + G-6-P + 6-P-G + 6-P-G f NADPH NADI’H NADPH
NADPf f 5 mg/ml NAD+ NADPf NADP+ + 0.25 mgjml NAD+ NADP’ + 5 n&ml NADf + 0.25 mg/ml NAD+
8. 5 mg/ml NAD+ 9. 0.25 mgjml NADf IO.
38 Albino
33 August
36 August
7.3 x!I0.8 1.0+0.1 -
4.7 i 0.4 0.3 * 0.04 -
7.4 t 1.0 0.4 i: 0.01 _c -
2.1 kO.4 -
0.5 t- 0.05 -
1.1 ct 0.1 -
4.2
3.9
5.9
6-P-G ==6-phosphogluconate.
DPSCUSSION The obiect of these studies was to test whether there was real significance in studying the metabolism of intact tissue slices. It was suggested that this type of investigation would have a place in the biochemical armamentarium if it could give information which was not readily obtainable by other techniques currently available. As a test case, the oxidation first of glutamate and then of glucose-(i-phosphate have been measured separately both in the intact section, namely in the presence of PVA, and in the relatively disrupted section (without the protective effect of PVA). When serial sections from the same tissue were allowed to oxidise both these substrates simultaneously, the oxidative activity was far greater than was the sum of the oxidation of each substrate, measured separately (Tables 1 and 2). However the efficiency of this activity was considerably enhanced by the protective effect of PVA (Tables 1 and 2). It seems reasonable to conclude, therefore, that the use of tissue sections may give more information on the inter-action between be obtained cytoplasmic and mitochondrial oxidative metabolism than could by other techniques. It was necessary to attempt to analyse the causes of the enhanced oxidation of these two substrates. The effect of various combinations of the components of the complete incubation medium were studied (Table 3). The addition of NAD+ to the incubation medium containing glucose-6-phosphate and NADP+ caused signifkantly enhanced oxidation, which at first sight might Experimental
Cell Resenrch 49
Intact
cirogencttion
37 AUgUSl
lo.oto.6 071-018 . .
in the presence
3x August
of 20 % PVrl.
19 Albino
5.8 ri: 0.4 0.9 2 0.2
-
38 Albino
38 August
25 Albino
39 Xibino
-
-
6.2 F 0.6 _-
-
663
tissue sections and metabolic inter-actions
0.4 F 0.04
-
-
7.9F0.5 -
7.2 f 0.6
0.4 & 0.1
1.5lO.l -
1.6-+0.4 _-_
0.9 iro.1
2.1t0.4 -
7.8
3.3
4.Q
4.9
._-
2.6i0.15
8.2 t 0.3
1.3 i: 0.5 1.6kO.4
0.4 5 0.06 -
1.7F0.2 -
-
0.3 i 0.07
0.9 -t 0.2
1.9
5.6
-
4.3
have been considered to be due to more efficient oxidation of glucose-6phosphate; however, addition of glutamate to this medium increased the deposition of formazan four- or five-fold (see Table 3, lines 3 and 1). This increase could not be explained by the effect of this concentration of glutamate and NADf alone (line 4, Table 3). Thus it seemed likely that there was a real enhancement of glutamate dehydrogenase activity in the presence of glucose-6-phosphate dehydrogenation. The increased activity shown by glucose-6-phosphate dehydrogenase in the presence of both co-enzymes (Table 3, lines 2, 3, and 8; also Tables 4 and 5), could have been due to an unsuspected requirement of this enzyme for both co-factors. However, when a similar requirement was found for 6-phosphogluconate dehydrogenase (Tables 4 and 5, lines 3, 4 and 9) it seemed possible that this effect could be due to an inter-action between the NADPH, generated by the dehydrogenase, and the KAD+. This inter-action n-as demonstrated (Tables 4 and 5, line 5 compared with lines 7 and 8; or line 6 compared with 7 and 9) most clearly when the incubation media contained PVA, that is when the tissue section remained intact [l, 21. These results seemed to indicate a transhydrogenation process. Some evidence for this transhydrogenation, and for the intra-mitochondrial nature of the hydrogen derived from this process, has been adduced from its effects on the histochemical reaction for cytochrome oxidase. In this reaction an indophenol dye is produced at the expense of oxidised cytochrome c, which becomes reduced in the process. It was argued that the content of oxidised cytochromc Experimental
Cell Research 49
R. G. Butcher and J. Ghayen
664
TABLE 5. The attempted de~o~lstration of ~ra~shydrogeRa~~on
in the absenceof PVA.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
G-6-P +NADP+ +5 mg/mI NAD* G-G-P + NADP+ 6-P-G t NADP* iO.25 mgjmi NAD+ 6-P-G i NADPMDPH it 5 mg/ml NAD” NADPH+0.25 mg/ml NAD+ NADPH 5 mg/ml WdY 0.25 mg/ml MD*
c should be reduced if intra-mitochondrial hydrogen-transport occurred during the historhemical reaction for cytoehrome oxidase; this was demonstrated by the fact that succinate inhibited the production of the indophenol. Neither NAD+ nor NADPH, whether the latter was added as such or \vas generated from glucose-&phosphate dehydrogenase activity, affected the production of the dye. In marked contrast, indophenol production was inhibited when NADPH and NAD+ were added together to the intact tissue section (i.e. in PT’A) which was subjected to the reagents for the cytochrome osidase reaction. Thus it seems possible that the inter-action between NADPH and NAD+ may represent a true transhydrogenation between extra-mitochondrial NADPH and the intra-mitochondrial hydrogen-transport system. It is noteworthy that the clear ~Iemonstration of this effect requires a relatively intact tissue section. SUMMARY
It is suggested that [here are special advantages in studying the metabolic activity of whole intact tissue sections rather than that of homogenates. A critical test has been proposed: namely, whether inter-action between cytoplasmic and mitochondrial systems can be demonstrated. Consequently the metabolism of glucose-G-phosphate (cytoplasmic) and of glutamate jmitochondrial) has been investigated separately and when both substrates and cofactors are added sim~lltaneously. It has been found that intact E~perf~enfaf
Celt Research 49
Intnct
tissue sections crnd I~let~b~li~ inter-ffction~
665
sections, but not disrupted sections, do indeed show such interaction by a greatly enhanced production of hydrogen. A major part of this interaction has been shown to be due to transhydrogenation between the NADPH formed in the cytoplasm and NAP) +. Moreover it has been demonstrated that the NADH produced by this transhydrogenation becomes Iocated in the mitochondrial electron transport system and affects the cytochrome oxidase of the mitochondria. Hence it is concluded that tissue sections, if properly produced and processed, should have a significant part to play in metabolic biochemistry. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
ALTMANN,
F. P. and CHAYES,
.I., Satnre
207,
1205
(1965).
~
J. Roy. mieroscop. Sot. 85, 155 (1966). BITENSKY, L. Quart. J, microscop. Sei. 104, 193 (1963).
__ Ciba Symp. Lysosomes p. 362. ChurrhiIl Ltd., London (1963). BITENSKY, I,., BALDWIN, R. W. and CHAYEN, J., Bril. J. Carwer 14, 696 (1960). BUTCHER, R. G., ALTMANN, I:. P. and CHAYES, J., Proc. Roy microscop. Sot. 1, 215 (1966). BUTCHER, R. G. and CHAYEN, J., Biochem. J. 96, 53 P (1965). BUTCHER, R. G., DIENGDOII, J. V. and CHAYIZN, J., Quart. J. microscop. Sci. 105, 497 (1964). CHAYEN, J., in S. M. Russell and K. F. A. Ross feds), Cell Structure and its Interpretation. Arnold, London. In press. CIIAYEN, J. and BITENSKY, I.,., in E. E. Bittar (ed.).The Biological Basis of Medicine. Academic Press, London, 1968. CHI\YEN, J.. BITENSKY, L., BUTCHER, R. G. and POULTER. L. \I’.. A Guide to Practical Histochemistry. dliver & Boyd,‘Edinburgh, 1968. JONES, G. R. N., MAPLE, A. J., AVES, E. K., CHAYEN. J. and CUNNINGHARI, G. J.. , Xature 197. 4867 (1963). ' LYNCH, R., BITENSKY, L. and CHAYEN, J., J. Xog. mieroscop. Sot. 85, 213 (1965). SELIGMAN, A. M., in 1st. Int. Congress. Histocbem. Cytochem. Pergamon Press, Oxford, 1963. SILCOX, A. A., POULTER, L. VI;., BITENSKY, L. and CHAYEN, ,J., J. Roy. microscop. Sot. 84, 559 (1965).
Experimental
Cell Research 49
656
Experimental
1968 by Academic
Press Inc.
Cell Research 49, 656-665 (1968)
THE VALUE OF INTACT TISSUE SECTIONS FOR STUDYING METABOLIC INTER-ACTIONS BETWEEN THE CYTOPLASM AND MITOCHONDRIA R. G. BUTCHER Division
of Cellular
Biology,
and J. CHAYEN
The ~a~hilda and Terence Kennedy London W6, UK
Institute
of Rheumatology,
Received May 26, 1967l
The metabolic activities of mitochondria and of the other parts of the cytoplasm are most commonly studied by homogenizing the cells and separating the various fractions. It is well understood that these procedures may alter the activities of the parts so isolated. Moreover it is obvious that, after such procedures, any interactions between the parts can be calculated only by inference from the activities of the separate parts. This type of inference has an inherent assumption, namely that the metabolism of the cell is approximately the sum of the activities of the separate systems. Because this assumption, and these inter-actions, are of such importance, techniques have been devised which retain the normal spatial arrangement of cellular organelles, and the less particulate cytoplasm, even during biochemical study. Tissue is super-cooled [13] and sectioned at S-20 /J under such conditions that this super-cooled state is maintained [ 151. The sections are incubated in relatively conventional biochemical media; but to ensure that the non-denatured protoplasm is not solubilised during such incubation, a sufficient concentration of polyvinyl alcohol (PVA) must be incorporated into the media [1, 2 j. The validity of these procedures has been discussed fully (e.g. [9, 10, 111; also [3, 4] in relation to lysosomal integrity). Thus it is now possible to prepare sections of tissue in which the histological and even the cytological structure [5] is intact and to retain the tissue intact while it is tested for metabolic activity. Dehydrogenase activity can be measured quantitatively in such sections if neotetrazolium is used as the hydrogen-acceptor [14]; the deposit of reduced neotetrazolium (formazan) is eluted and measured spectrophotometrically. Direct proportionality between the amount of dehydrogenation and the amount of formazan so produced has been demonstrated [la] and the results can then be expressed 1 Revised version received September 26, 1967. Experimental
Cell Research 49
Intact
tibiae sections and ~et~~ol~c inter-acfiorl~
657
against unit area, or unit tissue nitrogen, or wit amount of tissue nucleic acid [?, $11. The crucial test of whether such procedures have a signi~cant place in biochemistry is whether they can give direct information concerning interactions between different parts of cells, whereas such ~rlter-actions might be espected to be distorted or to be derivable only by inference by conventional biochemical procedures. Of especial interest has been the apparent need for oxidation associated with the pentose shunt in maintaining the integrity of the more soluble parts of these sections [12]. Therefore it seemed that a rigorous test would be to examine a l~ito~holldrial system, namefy the oxidation of glutamate, and to see if this was affected by cytoplasmic oxidation. Moreover, the results with P\rA meant that any interacting systems could be investigated when in the iutact section (i.e. in the presence of WA) and compared to \vhat happens in the relatively homogenisetf tissue (namely the same type of system but in the absence of P\‘A). MATERIALS
AND
METHODS
Two strains of rats were used: albino Wistar rats and inbred August rats. They were killed by nitrogen and small pieces (about 0.5 cma) of their livers were chilled in hexane at about - 70°C. Sections (B-14 ,u) were cut at an ambient temperature of between - 25” and - 30°C with a chilled knife [ll, 15]; they were partially dried by the fact that they were picked off the knife on to a warm slide [Ill. At laboratory temperature, a ring of Perspex was sealed around each section by means of a film of Yaseline [6, II]. A measured volume of incubation medium, previously equilibrated to 37X, was put into the ring; after a standard time the medium and the ring were removed, the sect.ions were rinsed in distilled water and dried. The area of the section was measured by planimetry of the image, projected through a photographic enlarger. The Pormazan in the section was then eluted into 0.25 ml of 10 per cent nheptanol in sgm-tetrachloroethane 1121 and measured in microcells in a Hilger spectrophotometer at 559 m/J. The amount of formazan was determined by reference to a standard calibration graph and was related to unit area (by planimetry) and thus to unit volume of tissue assuming the thickness of the section to be that shown on the setting of the microtome. (This assumption was made on the basis of previous studies which related ~lominal thickness to amount of tissue nitrogen [7].) For each (determination four sections were measured and the result expressed as the mean value and standard deviation. The latter also constitutes a check on the conformity of thickness. All incubation media contained 0.1 per cent Ileotetrazolium chloride (Universal Crop Protection) in 0.05 M glycyl-glycine buffer at pH 8.0. To some of these was added 20 per cent w/v of polyvinyl alcohol (PVA: polyviol MO5~1~0 from Bush, Beach & Segner Bayley). According to the system to be tested, the incubation medium contained all or various combinations of the following: 0.15 m&ml glucose-6phosphate (disodium salt; Boehringer); 0.25 m&ml N,4DP+ (Roehringer); 5 mg/rnX
658
R. G. Butcher and J. Chayen
(or in specified cases 0.25 mg/ml) NAD+ (Boehringer}; 17 mg/ml L-glutamic acid (sodium salt; Lights). In other media the glucose-6-phosphate and NADP+ were replaced by 0.25 mg/ml NADPH (Boehringer). In some media the glucose-6-phosphate was replaced by 0.15 mg/ml 6-phosphogluconate (trisodium salt; Wessex). Cytochrome oxidase was demonstrated by the method of Butcher et crl. [8] at pH 8.0 and 37°C. In some experiments 20 per cent PVA was added to this medium. RESULTS
Comparison of g~l~tarnate and ~la~ose-6-phosphate studied singly or in combination
dehydrogenation
when
In these experiments the concentration of glucose-6-phosphate and of NADP+ has been kept to l/l0 optimal in an attempt to clarify the mechanism by which this pen&se-shunt system inter-acts with the glutamate dehydrogenase system. Consequently the activity shown by the tissue when incubated in this medium was consistently low (line 2, Tables 1 and 2). In most experiments the amount of oxidation of glutamate was enhanced if tested in the presenee of PVA (compare line 3, Tables 1 and 2). The sum of these two separate deh~drogenase systems (line 2 +line 3) never equalled the activity shown when serial sections from the same block of tissue were incubated in a medium containing the same concentration of both substrates and both co-enzymes (line 1, Tables 1 and 2). The difference between the total activity and the sum of the separate activities (line 4, i.e. lines l-(2 +3) was always greater when tested in the presence of PVA than in its absence (comparison of line 4 in Table 1 against line 4 in Table 2). This is especially apparent when the results from the same animal are compared (line 4, Tables 1 and 2, e.g. Expt Nos 15 and 33). TABLE
1. The actiuities of glucose-6-phosphate and glutamate when studied singly and in combination. Incubations
in the presence
dehydrogenases
of PVA.
pg Formazan/mm3/h Experiment Strain
1. G-6-P
+ NADPf
2. G-6-P
+ iXADP+
3. GLUT
+ NADf
number of rat . . .
+ GLUT
.. .
10 Albino
-t NAT)+
17.5 ci: 0.9 0.6 i: 0.04 3.4io.3
4. l-(2+3) G-6-P
13.5 = glucose-6-phosphate.
Experimental
GLUT
Cell Research 49
= glutamate.
38 Albino
33 August
36 August
3s August
27.3k 1.3 0.6 IO.06 2.0f0.15
2&I&0.6
27.153.9
32.2k1.6
24.0+1.:
1.OiO.l 12.1k1.7
lO.SkO.4
17.2k2.2
0.9 ILo.: 13.030.:
24.7
15.3
16.2
14.6
10.1
15 Albino
0.3 io.04
0.4 rf: 0.01
Intact TABLE
tissue sections and metabolic inter-actions
2. The activities of glucose-6-phosphate and glutamate when studied singly and in combination. Incubations
659 dehydrogenases
in the absence of PVA.
pg Formazan/mm3/h Experiment number . . . Strain of rat . 1. G-6-P + NADP+ GLUT + NAD+
1 Albino
2 Albino
3 Albino
10 Albino
15 Albino
16.9kO.9 1.2kO.l 3.8 io.3 11.9
11.9k0.4 1.7kO.l 3.0&0.1 7.2
13.6kO.7 1.4kO.2 2.2 +o.n 10.0
11.7F0.6 0.6iO.l 2.lkO.2 9.0
33 August
34 August
f
2. G-6-P + NADPf 3. GLIJT + Nr\D+ -1. 1 - (2 + 3)
The possibility
15.5 + 0.9 3.3 AZ0.3 2.4 k 0.2 9.8
of a transhydrogenation
phenomenon
9.3f0.7 9.3kO.6 0.3 + 0.06 0.2 &- 0.02 1.3iO.l l.Ot-0.12 8.0 7.8
in the combined system
IVith two substrates and two co-enzymes there were 14 possible permutations which could be contributing to this combined activity. Many have been tested for some of the tissues (Tables 4 and 5) but all have been tested at the same time on one tissue, namely that taken from animal No. 33. To do this, sections were incubated in the basic medium, either with or without PVA, to which one, two three or all four of the factors (either substrate or co-enzyme) were added and the amount of dehydrogenation produced from the given selection of factors was measured (Table 3). The only unusual combination to produce a significant difference was the addition of NAD+ to the normal NADP+-glucose-6-phosphate medium (compare line 3 with line 2, Table 3). This enhancement of dehydrogenation over that produced from either cofactor or from glucose-6-phosphate with either cofactor, could have two possible explanations. One would be that glucose-B-phosphate dehydrogenase requires both co-enzymes for full activity in such sections; the other would be that there is inter-action between NAD+ and the NADPH generated by the action of glucose-B-phosphate dehydrogenase. To test this, NADPH was substituted for glucose-6-phosphate and NL4DP+, in the presence of NAD+. The presence of NADPH together with NAD+ produced considerably more formazan than did either tested alone (comparison of line 7 and line 8 with line 5 in Table 4; also line 7 and line 9 with line 6 in Tables 4 and 5). In another experiment 6-phosphogluconate was substituted for glucose-6phosphate, at equimolar concentration, and this too showed the enhanced effect of having both co-enzymes present, so indicating that it was predominantly an inter-action of NPlDPH with NAD+, irrespective of how the Experimental
Celt Research 49
N. G. Butcher
660 TABLE
and J. Chayen
3. The effect of the various combinations of substrates and coenzymes on disrupted (no PVA) and intact (with 20 y0 PVA) tissue sections ,ug Formazan/mm3/h No PVA 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 23. 14. 15.
G-6-P + NADP’ t GLUT f NAD+ G-G-P I- NADPf G-6-P + NADPI’ i NAD+ GLUT + NAD’GLUT + SAD+, -t- NADP” G-6-P -6 GLI:T i- NAD’ G-6-P + GLIJT i NADP+ G-6-P+ NADf GLI’T/ i- NADP” XADP-‘iSAD-’ G-6-P + GLUT G-6-P SADP’ GLUT NAD+
9.3 *o.i 0.3 iO.06 2.0+0.14
1.0+0.12 1.1 io.03 l.Z&O.Z 1.1+-0.01 0.3 +0.03 0 0.3 & 0.01 0 0 0 0 0.3 20.03
lndophenol
Reduced C,Aachromca,
Cytochrame c
Oxidose (0)
Fig. 1. Eqerimenfal
Cell Research 49
r+
Ox,d,ied Cytochrome
c ’
%’
20 % PTA 27.1 0.3 3.7 10.6 9.3 11.2 1.6 0.5 0 0.5 0 0 0 0 0.5
k3.9 +0.04 kO.4 io.4 10.3 10.3 lo.1 *o.os * 0.05
+0.05
Intact
tissue and sections ll~efaboli~ infer-~~fio~s
661
former was supplied to the system (comparison of lines 4 and 9 with line 3, Tables 4 and 5). The extent of the “transhydrogenation” in the presence and absence of PVA, is given in line 10 in Tables 4 and 5; only in the former, i.e. in the intact tissue, is this el’fert appreciable.
If these results indicate the existence of a true transhydrogenation mechanism then it would be valuable to have a way of testing whether the hydrogen, derived from the pentose-shunt, can be shown inside the mitochondria. An attempt to do this has been made by examining the cytochemical cytochrome oxidase activity shown by these sections. This activity depends on the presence of oxidised cytochrome c: Fig. 1. It therefore seemed reasonable to test whether mitochondrial dehgdrogenase activity could reduce the cytochrome c sufficiently to abolish this cytochemical reaction. To investigate this, serial sections were incubated (,a) in the medium for demonstrating cytochrome oxidase activity alone, or (b) in the same medium to \\-hich enough succinate (0.05 M) was added as would be required for the histochemical demonstration of surcinate dehydrogenase. It was found that the presence of this con~entratio~l of succinate completely inhibite~l the histothemiral demonstration of cytochrome oxidase. In marked contrast, the addition of glucose-g-phosphate and KADP+ to the normal incubation medium for cytochrome oxidase, whether this contained PVA or not, had no effect on the production of the indophenol. Since these results indicated that intra-mitochondrial hydrogen-transport could inhibit the histochemical demonstration of cytoehrome oxidase activity, assurnabiy h;v ll~aintaiiling the ~~t(~chrome c in the reduced state, it seemed reasonable to test the apparent transhy[lrogenati(~ll process to see \\hether it contributed to this mitochondrial pathway. Consequentiy tests were made, in the presence oC 20 per vent PVA, of the elects of adding 0.25 mg/ml NADPH (3 x 1W4 N) and 0.25 mg/ml NAD+ (3.8 x 10V4 M), either singly or together, to the incubation medium for cytochrome oxidase. Each separately had no effect whereas when they were added together practically no stain for cytothrome oxidase was obtained, that is, there was no l~ro(l~l~tion of indophenol. This effect was found only in the presence of PVA; when glucose-6-phosphate and both NADP+ and KAD+ were added to the incubation medimn for cptochrome oxidase but without the addition of PVA, these additional ingredients had no efTect on the normal production of the indophenol. Exi~erirn~rli~l
Ceil Research 49
R. G. Butcher and J. Chayen
662
pg Formazan/mm3/h Experiment number . . . Strain of rat ,.. 1. 2. 3. 4. 5. 6. 7.
G-6-P + G-6-P + 6-P-G + 6-P-G f NADPH NADI’H NADPH
NADPf f 5 mg/ml NAD+ NADPf NADP+ + 0.25 mgjml NAD+ NADP’ + 5 n&ml NADf + 0.25 mg/ml NAD+
8. 5 mg/ml NAD+ 9. 0.25 mgjml NADf IO.
38 Albino
33 August
36 August
7.3 x!I0.8 1.0+0.1 -
4.7 i 0.4 0.3 * 0.04 -
7.4 t 1.0 0.4 i: 0.01 _c -
2.1 kO.4 -
0.5 t- 0.05 -
1.1 ct 0.1 -
4.2
3.9
5.9
6-P-G ==6-phosphogluconate.
DPSCUSSION The obiect of these studies was to test whether there was real significance in studying the metabolism of intact tissue slices. It was suggested that this type of investigation would have a place in the biochemical armamentarium if it could give information which was not readily obtainable by other techniques currently available. As a test case, the oxidation first of glutamate and then of glucose-(i-phosphate have been measured separately both in the intact section, namely in the presence of PVA, and in the relatively disrupted section (without the protective effect of PVA). When serial sections from the same tissue were allowed to oxidise both these substrates simultaneously, the oxidative activity was far greater than was the sum of the oxidation of each substrate, measured separately (Tables 1 and 2). However the efficiency of this activity was considerably enhanced by the protective effect of PVA (Tables 1 and 2). It seems reasonable to conclude, therefore, that the use of tissue sections may give more information on the inter-action between be obtained cytoplasmic and mitochondrial oxidative metabolism than could by other techniques. It was necessary to attempt to analyse the causes of the enhanced oxidation of these two substrates. The effect of various combinations of the components of the complete incubation medium were studied (Table 3). The addition of NAD+ to the incubation medium containing glucose-6-phosphate and NADP+ caused signifkantly enhanced oxidation, which at first sight might Experimental
Cell Resenrch 49
Intact
cirogencttion
37 AUgUSl
lo.oto.6 071-018 . .
in the presence
3x August
of 20 % PVrl.
19 Albino
5.8 ri: 0.4 0.9 2 0.2
-
38 Albino
38 August
25 Albino
39 Xibino
-
-
6.2 F 0.6 _-
-
663
tissue sections and metabolic inter-actions
0.4 F 0.04
-
-
7.9F0.5 -
7.2 f 0.6
0.4 & 0.1
1.5lO.l -
1.6-+0.4 _-_
0.9 iro.1
2.1t0.4 -
7.8
3.3
4.Q
4.9
._-
2.6i0.15
8.2 t 0.3
1.3 i: 0.5 1.6kO.4
0.4 5 0.06 -
1.7F0.2 -
-
0.3 i 0.07
0.9 -t 0.2
1.9
5.6
-
4.3
have been considered to be due to more efficient oxidation of glucose-6phosphate; however, addition of glutamate to this medium increased the deposition of formazan four- or five-fold (see Table 3, lines 3 and 1). This increase could not be explained by the effect of this concentration of glutamate and NADf alone (line 4, Table 3). Thus it seemed likely that there was a real enhancement of glutamate dehydrogenase activity in the presence of glucose-6-phosphate dehydrogenation. The increased activity shown by glucose-6-phosphate dehydrogenase in the presence of both co-enzymes (Table 3, lines 2, 3, and 8; also Tables 4 and 5), could have been due to an unsuspected requirement of this enzyme for both co-factors. However, when a similar requirement was found for 6-phosphogluconate dehydrogenase (Tables 4 and 5, lines 3, 4 and 9) it seemed possible that this effect could be due to an inter-action between the NADPH, generated by the dehydrogenase, and the KAD+. This inter-action n-as demonstrated (Tables 4 and 5, line 5 compared with lines 7 and 8; or line 6 compared with 7 and 9) most clearly when the incubation media contained PVA, that is when the tissue section remained intact [l, 21. These results seemed to indicate a transhydrogenation process. Some evidence for this transhydrogenation, and for the intra-mitochondrial nature of the hydrogen derived from this process, has been adduced from its effects on the histochemical reaction for cytochrome oxidase. In this reaction an indophenol dye is produced at the expense of oxidised cytochrome c, which becomes reduced in the process. It was argued that the content of oxidised cytochromc Experimental
Cell Research 49
R. G. Butcher and J. Ghayen
664
TABLE 5. The attempted de~o~lstration of ~ra~shydrogeRa~~on
in the absenceof PVA.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
G-6-P +NADP+ +5 mg/mI NAD* G-G-P + NADP+ 6-P-G t NADP* iO.25 mgjmi NAD+ 6-P-G i NADPMDPH it 5 mg/ml NAD” NADPH+0.25 mg/ml NAD+ NADPH 5 mg/ml WdY 0.25 mg/ml MD*
c should be reduced if intra-mitochondrial hydrogen-transport occurred during the historhemical reaction for cytoehrome oxidase; this was demonstrated by the fact that succinate inhibited the production of the indophenol. Neither NAD+ nor NADPH, whether the latter was added as such or \vas generated from glucose-&phosphate dehydrogenase activity, affected the production of the dye. In marked contrast, indophenol production was inhibited when NADPH and NAD+ were added together to the intact tissue section (i.e. in PT’A) which was subjected to the reagents for the cytochrome osidase reaction. Thus it seems possible that the inter-action between NADPH and NAD+ may represent a true transhydrogenation between extra-mitochondrial NADPH and the intra-mitochondrial hydrogen-transport system. It is noteworthy that the clear ~Iemonstration of this effect requires a relatively intact tissue section. SUMMARY
It is suggested that [here are special advantages in studying the metabolic activity of whole intact tissue sections rather than that of homogenates. A critical test has been proposed: namely, whether inter-action between cytoplasmic and mitochondrial systems can be demonstrated. Consequently the metabolism of glucose-G-phosphate (cytoplasmic) and of glutamate jmitochondrial) has been investigated separately and when both substrates and cofactors are added sim~lltaneously. It has been found that intact E~perf~enfaf
Celt Research 49
Intnct
tissue sections crnd I~let~b~li~ inter-ffction~
665
sections, but not disrupted sections, do indeed show such interaction by a greatly enhanced production of hydrogen. A major part of this interaction has been shown to be due to transhydrogenation between the NADPH formed in the cytoplasm and NAP) +. Moreover it has been demonstrated that the NADH produced by this transhydrogenation becomes Iocated in the mitochondrial electron transport system and affects the cytochrome oxidase of the mitochondria. Hence it is concluded that tissue sections, if properly produced and processed, should have a significant part to play in metabolic biochemistry. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
ALTMANN,
F. P. and CHAYES,
.I., Satnre
207,
1205
(1965).
~
J. Roy. mieroscop. Sot. 85, 155 (1966). BITENSKY, L. Quart. J, microscop. Sei. 104, 193 (1963).
__ Ciba Symp. Lysosomes p. 362. ChurrhiIl Ltd., London (1963). BITENSKY, I,., BALDWIN, R. W. and CHAYEN, J., Bril. J. Carwer 14, 696 (1960). BUTCHER, R. G., ALTMANN, I:. P. and CHAYES, J., Proc. Roy microscop. Sot. 1, 215 (1966). BUTCHER, R. G. and CHAYEN, J., Biochem. J. 96, 53 P (1965). BUTCHER, R. G., DIENGDOII, J. V. and CHAYIZN, J., Quart. J. microscop. Sci. 105, 497 (1964). CHAYEN, J., in S. M. Russell and K. F. A. Ross feds), Cell Structure and its Interpretation. Arnold, London. In press. CIIAYEN, J. and BITENSKY, I.,., in E. E. Bittar (ed.).The Biological Basis of Medicine. Academic Press, London, 1968. CHI\YEN, J.. BITENSKY, L., BUTCHER, R. G. and POULTER. L. \I’.. A Guide to Practical Histochemistry. dliver & Boyd,‘Edinburgh, 1968. JONES, G. R. N., MAPLE, A. J., AVES, E. K., CHAYEN. J. and CUNNINGHARI, G. J.. , Xature 197. 4867 (1963). ' LYNCH, R., BITENSKY, L. and CHAYEN, J., J. Xog. mieroscop. Sot. 85, 213 (1965). SELIGMAN, A. M., in 1st. Int. Congress. Histocbem. Cytochem. Pergamon Press, Oxford, 1963. SILCOX, A. A., POULTER, L. VI;., BITENSKY, L. and CHAYEN, ,J., J. Roy. microscop. Sot. 84, 559 (1965).
Experimental
Cell Research 49