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Inhibition of mitochondrial protein synthesis by nitrofurantoin in rat and goat liver
470
Short ~ommullications
Howwr,whzrz theijsosomes were treated with both enzyme and prrdnisolonc for 7 hr or by 5 x IO ’ M prednisolone for 60 min followd by PLC for 60 min the steroid has competed successfully against the enzyme for the phospholipid sites as is shown by lower levels of enzyme in the supernatant. In a parallel set of experiments lysosomal suspensions Mere pretreated rcparatcl> for 60 min at 37 C‘ withoortisol,cortiso~~~.dex;~rnethasone.prcdnisonc.prcd~~isolot~c;~nd trialllclnololle;~t a tinal concentration of 5 x I()-’ M. Then TNBS has added to a linal concentration of I mM and incubation continued for a further 60 min. When compared to controls no stabilization against the Iytic action of TNBS was found with an) stei-aid tcstcd.
3~kilon,/c,rl~~r,lerlr One of us (,4.M.S.) wishes to thank the 14edtcal Research (‘ounuil for a postgt-aduatc training award during the tenure of which this v.ork ux’r done at the llniversit~ of Bath.
ANI)KI:M’ M. Sshto~s
ROUALIIJ. ANCILI.
REFERENCES I. A. D. BA\GHAM%M. M. STANDISHand J. C. WATU~S. J. ,Vo/ee. Bioi. 13, 23X (1965). 2. D. A. Lt.%%. A. M. SVMONSand R. J. ANCIU. J. Phurrn. Phtrvrnir~. 22, 902 (1970).
Inhibition of mitochondrial
protein synthesis by nitrofurantoin
in rat and goat liver
THE MECHANISM of action of nitrofurantoin has not been inwxtigated thoroughly although this and related compounds have been known for almost 43 yr.’ Roschenthaler 01 u[. examined the effect of nitrofurantoin upon RNA and protein synthesis in E. co/i Klz by using the /i’-galactosidase system and concluded that the inhibition of the enzyme synthesis could he due either (i) to a decreased pool of nucleotide triphosphates b\ interference with the energq metabolism, or (ii) to disturbances of the cell membrane or (iii1 to a more direct action on the protein synthesis apparatus.’ It is now well established that mitochondria possess their own protein synthesizing machinery’ ’ and some features of the mito~hondrial protein synthesizing sqstcm resemble those of the bacterial system.“,;‘ We present the results of a stud? of the effect of nitrofu~~ntoin on the inc{~rpor~ition of ‘“C‘-vaiine into proteins 11~intact niitachondri:i 15olatcd from rat and goat tiver.
471
Short communications
TAI~LI I.Ett1r-rOF N,TRO~,RAiiTO,NAX,>OTHlR INHIHITOKSON 'THtI~CORPoRATtoN OF L-VALIW-~~-'J< INTO PROTLINSBY ISOLATED MITOCHONDRIA i-ROMRAT ANI)GOAT LlVtR Incorporation (counts, min per mg protein) S]
Rat libcr
stem
Complete Complete ~ ATP and its gcncrating ~!s~cm Complctc + RNa\c (20 /~g) Complete + qcloheximide (75 A() Complctc + chlol-amphenicol (SO l(g) Complctc + NFT (IO l(g) Complctc + NFT (30 llg) Complctc - ATP and its gcncrating system + NFT (10 beg, NFT: Stands for nitrofurantoin. Rewlts arc the averages of five experiments
4250 4100 1260 4090 x50 3200 380
4800 4760 4780 4700 990 2480 500
i_ 370 * 390 * 400 + 380 +_ 75 * I60 * 30
2180 * 130
uith
* * & * * * *
-WI 330 420 310 70 230 50
2410 + ?I10
&SD.
prepared according to the method described by Dube et (separately) with Potter-Elvehjem homogenizer in 8 vol. of ice-cold medium A (0.25 M sucrose. 0.05 M Tris-HCl buffer. pH 7.4 and 0,025 M potassium phosphate buffer. pH 7.4). The homogenate was ccntrifugcd at 1000 6, for 10 min at 0 The supernatant fluid \\;I!, finally ccntrifugcd at 10.000 y to obtain the mitochondrial fraction. The 10,000 y pellet thus separatcd ~a> \\ashrd tmicc \\ith cold medium A. Isolated mitochondria were further purified on a discontinuous sucrose gradient according to the method of Richter and Lipmann’ as described elsewhere.“’ The complete incubation system contained 1Itmole of ATP. 3 fitmoles phosphoenolpyruvate. 5 pmoles of MgCI,. 20 icmoles of sucrose. 50 btmoles of Tris- HCI buffer (pH 7.4). 20 /imoles of potassium phosphate butrUr (pH 7.3~. r-Valine-U-‘sC (counts min 1.10 x IO-‘). specific radioacticitl (30 mCi:m-moles) and 44.5 mg of mitochondrial protein. The total volume of the incubation mixture was I ml. The incubation was carried out aerobically for 2 hr at 37 with constant shaking. The incubation was stopped by the addition of 0.3 ml of 30” 0 TCA. For the measurement of radioactivit\i. protein was processed according to the method of Stachiewicr and Quastel as described by Dube L’Tul. ” Protein content qf the mitochondrial fraction was measured according to the method of Lowry 01 trl.” The data given in Table I indicate that mitochondria from goat and rat liver can incorporate labelled valine into proteins. The incorporation is resistant to RNase which clearly rules out the doubt about the intactness of mitochondrin.‘” Again. the incorporation is actively taking place even in the absence of externail) added ATP and it5 gcncrating sqstcm indicating the prcscncc of sizable nucleotide triphosphatc pool within the mltochondria. Cyclohcximide. an inhibitor of eukaryotic protein synthesis. has no effect on mitochondrial protein synthesis in the present system indicating the absence of microsomal contamination. but chloramphrnicol inhibits the incorporation of valine to a great extent:’ ’ I4 Nitrofurantoin inhibits lahelled valine incorporation cvcn at a concentration as low as 10 &ml and a 90 per cent inhibition takes place at a concentration of 30 l(g ml. The inhibitor) efiect of nitrofurantoin in both the systems has been found cvcn in the ahscncc of cxternall! added ATP and its generating system. From the present stud? it ma) be concluded that the inhibition of incorporation of “C-amino acids into mitochondrial protclns is due (il to a direct action on the protein synthetic apparatus or, (ii) to a disturbance of mitochondrial membrane. but not due to a decreased nucleotide triphosphates pool by the interference with wergy metabolism. Furthe]- studies are required to reveal the actual locii where nitrofurantoin acts. Mitochondria
from goat and rat liker uere
LII.’ Liver from both the sources was homogenircd
.3~i\rlo~~/cl/~~t~ll~,/~r~~Thanks are due to University assistance.
Grants
Commission.
New Delhi.
India
for financial
BISWENUL B.GOSWAMI SVAMALIMACHAKRARARTI PRABIRBHATTACHARYYA SANDIP N. SINHA BIMAL K.Rol P~.RNIW RA>CHOI!IIHIRI DIPAK K. Dt 15,
472
Short communications
REFERENCES
I, K. Mu
IIA
and H. K. RI:UXNOOKF. Proqr. Moe/. C/IF~I.5, 320 (1967).
2. K. ROSCHLMTHALLK.P. KIXIXI K. P. HI.KKLI(.H and J. IC,IN)ICLVI. Z/I/. L&/Xi. I -li)~. OIY/. 215, 303 I 1970). 3. M. ASH\VI,LL and J. S. Woati. 41~1. Rc~. B~oc~/wu. 39, 15 I ( 1970). 4. W. E. BAI<\I rI. D. H. BKOWZI and J. L. CPI.I 1. Prw. UCI(JI. I~od. SC!. I ..S..I. 57, 1775 (lYh71. 5. H. K. DAS. S. K. C’HATTIICJU and S. C. RI)), .I. iliol. (‘/x,uI. 243, 40x9 (1964). 6. J. P. Lbrs and E. B. KI:L.Lt:a. Biochc~~. Elio[~h~,s.K(a,\. C‘~I~IIIII~U. 40. 416 (1970). 7. J. Lx,ri\s-L~ NAaI) and F. LIPMANN. -Ir?rl. K~II,.Bioc,l~o~~.40. 433 (I97 I ). 8. D. K. Dt’trl. S. CHAI(KAHARTI. P. BHATTA(‘HAll\rh. B. B. GOS\VAMl mid s. c'.Ro>. &oc./w,!I. P/l~~).~lltu. 22,659(19731 9. D. Rlc.II rt a and F.
LIPMANU.
Bioc~hcuri\rr~~
9,
5065
(19701.
IO. B. B. GOS\VA’MI.S. CHAliaAaAaTI. D. K. Dtlnl and S. C‘. Rok. Hio&rr?i. .1.. 134. Xl5 (IY7.~l. I I. D. K. DUI~I S. C‘HAKKARARTIand S. C. Rol. Ctrnc,<~ 29, IS75 ( 1972). 12. 0. H. Loway, N. J. RoShllKOIIGH. A. 1. FARI< and R. J. RA\II)ALI. ./. Ilir~/. (‘/?c,ii~. 193. 265 (I951 1.
13. R. F. SWANSON. ,Vtrr~~~. Lo/~t/. 231, ?I (I97 I ). 14. S. C‘~iAtc!uisAKri. D. K. Dr ~~~and S. (‘. Ro\. B!or+~~rr. .I. 128. 461 (lY7?1.
The passage of &aminolaevulinic
acid across the blood brain barrier of the rat: effect of ethanol
ONE OF the distinctive features of acute Intermittent porphqrla (AIP) is the cxcessivc urinary crrcrctlon of certain porphyrins and their precursors, &aminolacvulinic acid (ALA) and porphohilinogcn (PBG). This is a result of the genetically mediated increase in porphyrin hiosynthcsis characteristic of the discnse One result of this elevated biosynthesis is that there is an associated incrcasc in the plasma concentration of ALA. ALA is virtually undetectable in the plasma of normal suhlccts. but ;I Ic~cl of 24 /ig ml ha\ bc‘cn ircported in an acute porphyric attack. Similarly. normal ccrchrospinal flu~rl IC’SI-) contain\ no ALA. !cl !\LA can be detected in patients with A1P.l These ohservationg suggcsr that XI cIc\atc? pla\nxt co,,centrations ALA can pass the blood-brain barrier (BBB). thereby cntcring the brain tissue and C‘SF-. Thcrc is. however. conflicting evidence on this possibility. Thus. although Kramcl-L has indicated that ALA is readily taken up by brain tissue, Musyka3 has reported that the BBB is impermeable to ALA. The resolution of these conflicting reports is of importance since there is increasIng evidence that ALA may be an aetiological factor in the production of the clinical manifestations of the disease. .AIthough the purlfed porphyrins and porphohilinogen are pharamcologicall~ macti\c.’ II. 1 has hccn sho\\n to Inhlblt brain ATPase activity’ and membrane sodium transport ;,I tit~o.’ Ful-thcr. ALA cause\ hch;iG~~ural changes in mice“ and has a hlpotcnhlvc action in anacsthctiscd rats.> Such action\ 01‘ AL.,Z could hGlI some relationship to the fact that some 55 per cent of patients with AIP exhibit psqchologlcal symptom\: and post-mortem examination of porphyric patients has revealed central ncuropathj.’ Thcrc 1s also cotlvincing evidence that ALA can increase the susceptibility of expcrimcntal animals to drug induced convusions.’ Certain porphyrinogenic drugs are known to provoke attacks 111ruh~ccts with latent Alp.‘” Thcac drug\. which include ethanol, increase the activity of hcpatic ALA synthetase. the enzyme v.hich products ALA from glycine and succinyl-CoA. There is the possibility. thcrcfore. that the) precipitate porphyric attack by increasing the circulating level of ALA and thus. if ALA can pasr the BBB. the brain tissue level 01 ALA. Alternatively, they could cause the BBB to become permeable or more pcrmcahle to ALA Acute attacks of porphyria have been provoked hq an episode of over-indulgence in ethanol.’ ’ This cuperimcnt was designed therefore, to confirm whcthcr or not ALA could pass the BBB in no!-mal animal\ and 111 animals under acute ethanol intoxication.
Short ~ommullications
Howwr,whzrz theijsosomes were treated with both enzyme and prrdnisolonc for 7 hr or by 5 x IO ’ M prednisolone for 60 min followd by PLC for 60 min the steroid has competed successfully against the enzyme for the phospholipid sites as is shown by lower levels of enzyme in the supernatant. In a parallel set of experiments lysosomal suspensions Mere pretreated rcparatcl> for 60 min at 37 C‘ withoortisol,cortiso~~~.dex;~rnethasone.prcdnisonc.prcd~~isolot~c;~nd trialllclnololle;~t a tinal concentration of 5 x I()-’ M. Then TNBS has added to a linal concentration of I mM and incubation continued for a further 60 min. When compared to controls no stabilization against the Iytic action of TNBS was found with an) stei-aid tcstcd.
3~kilon,/c,rl~~r,lerlr One of us (,4.M.S.) wishes to thank the 14edtcal Research (‘ounuil for a postgt-aduatc training award during the tenure of which this v.ork ux’r done at the llniversit~ of Bath.
ANI)KI:M’ M. Sshto~s
ROUALIIJ. ANCILI.
REFERENCES I. A. D. BA\GHAM%M. M. STANDISHand J. C. WATU~S. J. ,Vo/ee. Bioi. 13, 23X (1965). 2. D. A. Lt.%%. A. M. SVMONSand R. J. ANCIU. J. Phurrn. Phtrvrnir~. 22, 902 (1970).
Inhibition of mitochondrial
protein synthesis by nitrofurantoin
in rat and goat liver
THE MECHANISM of action of nitrofurantoin has not been inwxtigated thoroughly although this and related compounds have been known for almost 43 yr.’ Roschenthaler 01 u[. examined the effect of nitrofurantoin upon RNA and protein synthesis in E. co/i Klz by using the /i’-galactosidase system and concluded that the inhibition of the enzyme synthesis could he due either (i) to a decreased pool of nucleotide triphosphates b\ interference with the energq metabolism, or (ii) to disturbances of the cell membrane or (iii1 to a more direct action on the protein synthesis apparatus.’ It is now well established that mitochondria possess their own protein synthesizing machinery’ ’ and some features of the mito~hondrial protein synthesizing sqstcm resemble those of the bacterial system.“,;‘ We present the results of a stud? of the effect of nitrofu~~ntoin on the inc{~rpor~ition of ‘“C‘-vaiine into proteins 11~intact niitachondri:i 15olatcd from rat and goat tiver.
471
Short communications
TAI~LI I.Ett1r-rOF N,TRO~,RAiiTO,NAX,>OTHlR INHIHITOKSON 'THtI~CORPoRATtoN OF L-VALIW-~~-'J< INTO PROTLINSBY ISOLATED MITOCHONDRIA i-ROMRAT ANI)GOAT LlVtR Incorporation (counts, min per mg protein) S]
Rat libcr
stem
Complete Complete ~ ATP and its gcncrating ~!s~cm Complctc + RNa\c (20 /~g) Complete + qcloheximide (75 A() Complctc + chlol-amphenicol (SO l(g) Complctc + NFT (IO l(g) Complctc + NFT (30 llg) Complctc - ATP and its gcncrating system + NFT (10 beg, NFT: Stands for nitrofurantoin. Rewlts arc the averages of five experiments
4250 4100 1260 4090 x50 3200 380
4800 4760 4780 4700 990 2480 500
i_ 370 * 390 * 400 + 380 +_ 75 * I60 * 30
2180 * 130
uith
* * & * * * *
-WI 330 420 310 70 230 50
2410 + ?I10
&SD.
prepared according to the method described by Dube et (separately) with Potter-Elvehjem homogenizer in 8 vol. of ice-cold medium A (0.25 M sucrose. 0.05 M Tris-HCl buffer. pH 7.4 and 0,025 M potassium phosphate buffer. pH 7.4). The homogenate was ccntrifugcd at 1000 6, for 10 min at 0 The supernatant fluid \\;I!, finally ccntrifugcd at 10.000 y to obtain the mitochondrial fraction. The 10,000 y pellet thus separatcd ~a> \\ashrd tmicc \\ith cold medium A. Isolated mitochondria were further purified on a discontinuous sucrose gradient according to the method of Richter and Lipmann’ as described elsewhere.“’ The complete incubation system contained 1Itmole of ATP. 3 fitmoles phosphoenolpyruvate. 5 pmoles of MgCI,. 20 icmoles of sucrose. 50 btmoles of Tris- HCI buffer (pH 7.4). 20 /imoles of potassium phosphate butrUr (pH 7.3~. r-Valine-U-‘sC (counts min 1.10 x IO-‘). specific radioacticitl (30 mCi:m-moles) and 44.5 mg of mitochondrial protein. The total volume of the incubation mixture was I ml. The incubation was carried out aerobically for 2 hr at 37 with constant shaking. The incubation was stopped by the addition of 0.3 ml of 30” 0 TCA. For the measurement of radioactivit\i. protein was processed according to the method of Stachiewicr and Quastel as described by Dube L’Tul. ” Protein content qf the mitochondrial fraction was measured according to the method of Lowry 01 trl.” The data given in Table I indicate that mitochondria from goat and rat liver can incorporate labelled valine into proteins. The incorporation is resistant to RNase which clearly rules out the doubt about the intactness of mitochondrin.‘” Again. the incorporation is actively taking place even in the absence of externail) added ATP and it5 gcncrating sqstcm indicating the prcscncc of sizable nucleotide triphosphatc pool within the mltochondria. Cyclohcximide. an inhibitor of eukaryotic protein synthesis. has no effect on mitochondrial protein synthesis in the present system indicating the absence of microsomal contamination. but chloramphrnicol inhibits the incorporation of valine to a great extent:’ ’ I4 Nitrofurantoin inhibits lahelled valine incorporation cvcn at a concentration as low as 10 &ml and a 90 per cent inhibition takes place at a concentration of 30 l(g ml. The inhibitor) efiect of nitrofurantoin in both the systems has been found cvcn in the ahscncc of cxternall! added ATP and its generating system. From the present stud? it ma) be concluded that the inhibition of incorporation of “C-amino acids into mitochondrial protclns is due (il to a direct action on the protein synthetic apparatus or, (ii) to a disturbance of mitochondrial membrane. but not due to a decreased nucleotide triphosphates pool by the interference with wergy metabolism. Furthe]- studies are required to reveal the actual locii where nitrofurantoin acts. Mitochondria
from goat and rat liker uere
LII.’ Liver from both the sources was homogenircd
.3~i\rlo~~/cl/~~t~ll~,/~r~~Thanks are due to University assistance.
Grants
Commission.
New Delhi.
India
for financial
BISWENUL B.GOSWAMI SVAMALIMACHAKRARARTI PRABIRBHATTACHARYYA SANDIP N. SINHA BIMAL K.Rol P~.RNIW RA>CHOI!IIHIRI DIPAK K. Dt 15,
472
Short communications
REFERENCES
I, K. Mu
IIA
and H. K. RI:UXNOOKF. Proqr. Moe/. C/IF~I.5, 320 (1967).
2. K. ROSCHLMTHALLK.P. KIXIXI K. P. HI.KKLI(.H and J. IC,IN)ICLVI. Z/I/. L&/Xi. I -li)~. OIY/. 215, 303 I 1970). 3. M. ASH\VI,LL and J. S. Woati. 41~1. Rc~. B~oc~/wu. 39, 15 I ( 1970). 4. W. E. BAI<\I rI. D. H. BKOWZI and J. L. CPI.I 1. Prw. UCI(JI. I~od. SC!. I ..S..I. 57, 1775 (lYh71. 5. H. K. DAS. S. K. C’HATTIICJU and S. C. RI)), .I. iliol. (‘/x,uI. 243, 40x9 (1964). 6. J. P. Lbrs and E. B. KI:L.Lt:a. Biochc~~. Elio[~h~,s.K(a,\. C‘~I~IIIII~U. 40. 416 (1970). 7. J. Lx,ri\s-L~ NAaI) and F. LIPMANN. -Ir?rl. K~II,.Bioc,l~o~~.40. 433 (I97 I ). 8. D. K. Dt’trl. S. CHAI(KAHARTI. P. BHATTA(‘HAll\rh. B. B. GOS\VAMl mid s. c'.Ro>. &oc./w,!I. P/l~~).~lltu. 22,659(19731 9. D. Rlc.II rt a and F.
LIPMANU.
Bioc~hcuri\rr~~
9,
5065
(19701.
IO. B. B. GOS\VA’MI.S. CHAliaAaAaTI. D. K. Dtlnl and S. C‘. Rok. Hio&rr?i. .1.. 134. Xl5 (IY7.~l. I I. D. K. DUI~I S. C‘HAKKARARTIand S. C. Rol. Ctrnc,<~ 29, IS75 ( 1972). 12. 0. H. Loway, N. J. RoShllKOIIGH. A. 1. FARI< and R. J. RA\II)ALI. ./. Ilir~/. (‘/?c,ii~. 193. 265 (I951 1.
13. R. F. SWANSON. ,Vtrr~~~. Lo/~t/. 231, ?I (I97 I ). 14. S. C‘~iAtc!uisAKri. D. K. Dr ~~~and S. (‘. Ro\. B!or+~~rr. .I. 128. 461 (lY7?1.
The passage of &aminolaevulinic
acid across the blood brain barrier of the rat: effect of ethanol
ONE OF the distinctive features of acute Intermittent porphqrla (AIP) is the cxcessivc urinary crrcrctlon of certain porphyrins and their precursors, &aminolacvulinic acid (ALA) and porphohilinogcn (PBG). This is a result of the genetically mediated increase in porphyrin hiosynthcsis characteristic of the discnse One result of this elevated biosynthesis is that there is an associated incrcasc in the plasma concentration of ALA. ALA is virtually undetectable in the plasma of normal suhlccts. but ;I Ic~cl of 24 /ig ml ha\ bc‘cn ircported in an acute porphyric attack. Similarly. normal ccrchrospinal flu~rl IC’SI-) contain\ no ALA. !cl !\LA can be detected in patients with A1P.l These ohservationg suggcsr that XI cIc\atc? pla\nxt co,,centrations ALA can pass the blood-brain barrier (BBB). thereby cntcring the brain tissue and C‘SF-. Thcrc is. however. conflicting evidence on this possibility. Thus. although Kramcl-L has indicated that ALA is readily taken up by brain tissue, Musyka3 has reported that the BBB is impermeable to ALA. The resolution of these conflicting reports is of importance since there is increasIng evidence that ALA may be an aetiological factor in the production of the clinical manifestations of the disease. .AIthough the purlfed porphyrins and porphohilinogen are pharamcologicall~ macti\c.’ II. 1 has hccn sho\\n to Inhlblt brain ATPase activity’ and membrane sodium transport ;,I tit~o.’ Ful-thcr. ALA cause\ hch;iG~~ural changes in mice“ and has a hlpotcnhlvc action in anacsthctiscd rats.> Such action\ 01‘ AL.,Z could hGlI some relationship to the fact that some 55 per cent of patients with AIP exhibit psqchologlcal symptom\: and post-mortem examination of porphyric patients has revealed central ncuropathj.’ Thcrc 1s also cotlvincing evidence that ALA can increase the susceptibility of expcrimcntal animals to drug induced convusions.’ Certain porphyrinogenic drugs are known to provoke attacks 111ruh~ccts with latent Alp.‘” Thcac drug\. which include ethanol, increase the activity of hcpatic ALA synthetase. the enzyme v.hich products ALA from glycine and succinyl-CoA. There is the possibility. thcrcfore. that the) precipitate porphyric attack by increasing the circulating level of ALA and thus. if ALA can pasr the BBB. the brain tissue level 01 ALA. Alternatively, they could cause the BBB to become permeable or more pcrmcahle to ALA Acute attacks of porphyria have been provoked hq an episode of over-indulgence in ethanol.’ ’ This cuperimcnt was designed therefore, to confirm whcthcr or not ALA could pass the BBB in no!-mal animal\ and 111 animals under acute ethanol intoxication.