isolation of rat brain mitochondria  all procedures involving 
  animals were approved by the children 's hospital of pittsburgh ( animal 
  protocol number chp upmc arcc 1805 ) and were in compliance with 
  principles of laboratory animal care and current laws of the united states . 
 
rat brain mitochondria were isolated from the cortex of adult sprague - dawley 
  rats . after removal , tissue was minced and homogenized in ice - cold isolation 
  buffer i , which contained 225 mm mannitol , 75 mm 
  sucrose , 5 mm hepes buffer ( ph adjusted to 7.3 with koh ) , 0.1 mg / ml 
  fatty acid - free bovine serum albumin , 1 mm tetrapotassium edta , and 
  12% percoll .
the homogenate thus obtained was carefully layered on the top of 
  a discontinuous gradient of percoll ( 24 and 42% ) prepared using the same 
  buffer . the preparation was then centrifuged at 16,000  g for 
  10 min .
the fraction containing the mitochondria located between 42 and 24% 
  percoll was carefully withdrawn by a syringe and washed from percoll twice by 
  pelleting in isolation buffer i. the resulting mitochondrial suspension was 
  diluted in isolation medium ii , which was similar to isolation buffer i , 
  except for the concentration of edta ( 0.1 mm ) and lack of albumin , 
  and spun down at 12,000  g for 10 min .
the deposit of 
  mitochondria was homogenized in isolation buffer ii at a final protein 
  concentration of 20 mg / ml and stored on ice until use .
the protein 
  concentration in the mitochondrial samples was determined using a protein 
  assay kit .
mitochondria prepared in this way were active for at least 
  56 h , as determined by their ability to maintain a stable transmembrane 
  potential in the presence of oxidizable substrates .
fluorescence measurements  measurements were performed in a 
  stirred cuvette mounted in a shimatzu rf-5301 spectrofluorimeter maintained at 
  37 c .
mitochondria ( 0.2 mg / ml protein ) were added to 1.5 ml of the basic 
  incubation medium , which contained 125 mm kcl , 5 mm 
  mgcl2 , 10 mm tris , 10 mm hepes ( ph adjusted 
  to a particular value between 6 and 8 with koh or hcl ) , 10 m 
  egta , 5 mm succinate or 5 mm glutamate , and 5 
  mm malate as oxidizable substrates . due to a tendency of 
  precipitation at alkaline ph in the presence of inorganic phosphate , 
  concentration of mgcl2 was reduced to 0.5 mm .
hydrogen peroxide  hydrogen peroxide was measured using a2 
  m concentration of the fluorescent amplex red dye in the 
  presence of 1 unit / ml horseradish peroxidase as previously described 
  ( 26 ) .
measurements were 
  carried out at excitation / emission wavelengths of 560 nm ( slit 1.5 nm)/590 nm 
  ( slit 3 nm ) , respectively .
h2o2 generation was 
  calibrated by constructing calibration curves using known 
  h2o2 concentrations in the standard incubation buffer 
  together with amplex red and horseradish peroxidase but without mitochondria 
  at all ph values 68 . at acidic ph ,
the correction coefficients calculated from the 
  titration curves for different medium ph were as follows : ph 6.0 , 0.68 ; ph 
  6.5 , 0.90 ; ph 7.0 , 1.00 ; ph 7.5 , 1.08 ; ph 8.0 , 1.10 .
these correction 
  coefficients were applied to compensate for a difference in 
  h2o2 reporting system response when determining rates of 
  ros release at a given ph .
mitochondrial 
  transmembrane potential ( m ) was estimated 
  using fluorescence quenching of the cationic dye safranin o that is 
  accumulated and quenched inside energized mitochondria .
the excitation 
  wavelength was 495 nm ( slit 3 nm ) and emission 586 nm ( slit 5 nm ) , and the dye 
  concentration used was 2.5 m 
  ( 11 ) .
respiration of mitochondria  respiration of mitochondria was 
  measured by a clark type oxygen electrode ( hansatech co. ; oxygen electrode 
  unit dw1 ) .
the water - jacketed chamber was maintained at 37 c and 
  constantly stirred with a magnetic stirring bar .
statistical analysis was performed using student 's 
  t test for comparison of two groups or one - way analysis of variance 
  with bonferroni 's correction for multiple comparison implemented in prism 4.0 
  software ( graphpad software , san diego , ca ) .
to study the ph - dependent component in the mechanism of reactive oxygen 
  species generation in mitochondria , we used three types of incubation media 
  with different impacts on the ph and electrical components of proton motive 
  force through the inner membrane : condition 1 , the medium without permeant 
  anions , such as pi ; condition 2 , the medium of condition 1 
  supplemented with pi ; condition 3 , the medium of condition 2 
  supplemented with nigericin .
rat brain mitochondria respiratory characteristics measured in condition 2 
  and ph variations from 6 to 8 with succinate or glutamate plus malate as 
  substrates are presented in fig . 
  1 .
these data show that our mitochondrial preparations are good in 
  coupling between respiration and phosphorylation in physiological range of ph . 
 
at ph 7 , the addition of oligomycin inhibits respiration , whereas the addition 
  of adp ( state 3 ( st 3 ) ) essentially stimulates it , so that the ratio 
  of respiration rates ( state 3/state oligomycin ( st 3/st 
  oligo ) ) was 5.6  0.6 ( n = 3 ) for succinate and 6.5 
   0.3 ( n = 6 ) for glutamate plus malate . at alkaline ph ,
adp 
  stimulated respiration to a smaller degree , possibly because the lack of 
  protons in the medium limits the process .
the first condition provides the maximal value of ph ( maximal ph in 
  mitochondrial matrix ( phin ) at a given ph of incubation medium 
  ( phout ) and minimal value of . pi co - transported with h ( or in exchange to 
  oh ) decreases ph ( and respectively increases  ) . 
 
therefore , the second condition stabilizes phin at a value less 
  alkaline compared with the first condition ; it provides the 
  h distribution between electrical and chemical 
  components similar to that in mitochondria of living cells .
figure 1.ph dependence of mitochondrial respiration in state 3 
  ( triangles ) , state 4 ( squares ) , and state 4 with added 
  oligomycin ( inverse triangles ) , with succinate ( a ) and 
  glutamate and malate ( b ) as substrates .
basic incubation medium 
  ( see  experimental procedures  ) supplemented with 1 mm 
  pi was used .
statistical analysis was as follows . a , rates 
  of respiration in state 3 taken at ph 8 were significantly different from that 
  at ph 67 ( n = 34 ; , p < 0.05 , 
  t test ) .
b , rates of respiration in state 3 taken at ph 8 
  were significantly different from that at ph 67.5 ; rates at ph 7 and 
  7.5 were significantly different from each other ( n = 6 ; 
  , p < 0.05 ; , p < 0.001 , 
  t test ) .
ph dependence of mitochondrial respiration in state 3 
  ( triangles ) , state 4 ( squares ) , and state 4 with added 
  oligomycin ( inverse triangles ) , with succinate ( a ) and 
  glutamate and malate ( b ) as substrates .
basic incubation medium 
  ( see  experimental procedures  ) supplemented with 1 mm 
  pi was used .
a , rates 
  of respiration in state 3 taken at ph 8 were significantly different from that 
  at ph 67 ( n = 34 ; , p < 0.05 , 
  t test ) .
b , rates of respiration in state 3 taken at ph 8 
  were significantly different from that at ph 67.5 ; rates at ph 7 and 
  7.5 were significantly different from each other ( n = 6 ; 
  , p < 0.05 ; , p < 0.001 , 
  t test ) .
nigericin as an h / k exchanger adjusts ph 
  according to the k gradient across the inner membrane . since 
  k is the major osmogenic component , mitochondrial volume normally 
  is stabilized when k concentrations inside and outside are the 
  same and therefore under the third condition phin = 
  phout . since the chemical component of h is 
  close to zero
,  could reach a higher value than under the other 
  conditions ( fig .
3a 
  ( squares ) shows that the rate of ros production under condition 1 is 
  3 times increased when the ph of media changed from 6 to 7 , whereas the levels 
  of membrane potential decreased ( fig . 
 
usually , high  is considered to be a factor 
  promoting ros production ( factor iii in the introduction ) .
here the situation 
  is controversial ; ros production is increased when  is decreased . 
  obviously , substrate and oxygen concentration ( factors ii and i ) remain the 
  same for all conditions ; therefore , medium ph is the only factor correlating 
  with increase of ros . at more alkaline ph ( 7.5 and 8) ,
4 
  demonstrates , the loss of membrane potential can be delayed by the presence in 
  the medium of the agents known to inhibit permeability transition pore 
  opening ; cyclosporine a together with adp and oligomycin .
this fact indicates 
  that the loss of membrane potential in this condition is due to permeability 
  transition pore opening .
additions at the time points indicated 
  by the arrows were as follows : nigericin , 100 nm ; fccp , 
  200 nm . nigericin increases membrane potential of mitochondria oxidizing 
  succinate at ph 68 .
additions at the time points indicated 
  by the arrows were as follows : nigericin , 100 nm ; fccp , 
  200 nm . in the presence of 1 mm of pi ( condition 2 ) , which 
  makes the conditions more physiological ,
the behavior of the analyzed system 
  is similar to that under condition 1 . as 
  fig .
3a 
  ( triangles ) shows , the rate of ros production steeply increased upon 
  increase of medium ph , reaching a maximum at ph 7.5 , while membrane potential 
  ( fig .
the decrease of ros production at ph 8 
  correlates with a slight decrease of respiration rate 
  ( fig .
3c , 
  triangles ) . when pi and nigericin were present in the medium ( condition 3 ) , 
  the rates of ros production also increased along with the medium ph increase 
  from 6 to 8 ( fig .
3c , 
  circles ) repeated the pattern when only pi was present 
  ( condition 2 ) , reaching maximum at ph 7.5 .
if ph = 0 , the whole 
  pattern remains qualitatively similar to that observed in the presence of 
  pi ( condition 2 ) .
the ph dependences presented in fig . 
  3 clearly show that ph itself , and not ph , is an important 
  determinant of ros production . as can be seen , at ph ranging from 6 to 7 , when 
  mitochondria do not undergo permeability transition , ros production under 
  condition 1 is higher than under conditions 2 or 3 , which could indicate an 
  important role of matrix ph .
the difference in ros production can be seen when 
  the conditions change in the course of the experiment , producing qualitatively 
  the same outcome ( i.e. the addition of pi or nigericin 
  always resulted in decrease of the ros generation rate despite the increase in 
   ( fig . 5 , a and 
  b ) because of trading the ph component of proton 
  motive force to the electric one ) . the difference in ros production between the three conditions presented in 
  fig .
in addition to the use of 
  pi and nigericin , application of the ionophores fccp and 
  valinomycin can be instrumental in manipulating matrix ph to study its role in 
  mitochondrial ros generation . both of these ionophores can decrease membrane 
  potential ; however , protonophore fccp acidifies the matrix by increasing 
  proton leak , whereas valinomycin causes increase of matrix ph by carrying 
  k into the matrix , decreasing  and , thus , facilitating 
  the proton ejection by the electron transport chain . to stress the 
  qualitatively different effects of these ionophores
, we applied a small 
  concentration of fccp that did not substantially change  and a 
  concentration of valinomycin that essentially depolarized mitochondria 
  ( fig .
6b shows , fccp 
  induced a dramatic decrease of ros production , wheras valinomycin temporarily 
  increased it .
figure 3.effect of medium ph on rate of ros release ( a ) , level of 
  membrane potential ( b ) , and rates of respiration of mitochondria 
  oxidizing succinate ( c ) . basic medium ( see  experimental 
  procedures  ) was used in all experiments with different 
  supplementations : condition 1 , basic medium alone ( squares ) ; 
  condition 2 , basic medium with 1 mm pi 
  ( triangles ) ; condition 3 , basic medium with 1 mm 
  pi and 100 nm nigericin ( circles ) . statistical 
  analysis
was as follows . a , ros rates taken at all conditions and all 
  ph values were significantly different from each other ( n = 
  310 , p < 0.05 ) , with an exception for conditions 2 and 3 at 
  ph 8 ( one - way analysis of variance test ) . within the same condition ,
ros rates 
  taken at each ph were significantly different from that at the adjacent ph 
  value ( n = 310 , p < 0.05 ) , with the exceptions of 
  the rates taken at ph 7.5 and 8 in condition 3 ( t test ) .
b , 
  the levels of membrane potential measured at ph 7.08.0 for condition 1 
  were significantly different from that in condition 2 or 3 ( n = 
  38 ; , p < 0.05 , analysis of variance test ) and 
  also from the membrane potential at ph 6.06.5 for the same condition 
  ( n = 3 ; + , p < 0.05 , t test ) .
c , at ph 
  8.0 , rates of respiration measured in conditions 2 and 3 were significantly 
  different from that in condition 1 ( n = 38 ; + , p < 
  0.05 , analysis of variance test ) . in condition 1 , rates at ph 8.0 were 
  significantly different from that at all other ph values ( n = 
  78 ; , p < 0.0001 , t test ) ; in 
  condition 3 , rates at ph 7.5 were significantly different from that at ph 6.0 
  and 6.5 ( n = 34 ; # , p < 0.05 , t 
  test ) . effect of medium ph on rate of ros release ( a ) , level of 
  membrane potential ( b ) , and rates of respiration of mitochondria 
  oxidizing succinate ( c ) .
basic medium ( see  experimental 
  procedures  ) was used in all experiments with different 
  supplementations : condition 1 , basic medium alone ( squares ) ; 
  condition 2 , basic medium with 1 mm pi 
  ( triangles ) ; condition 3 , basic medium with 1 mm 
  pi and 100 nm nigericin ( circles ) .
statistical 
  analysis was as follows . a , ros rates taken at all conditions and all 
  ph values were significantly different from each other ( n = 
  310 , p < 0.05 ) , with an exception for conditions 2 and 3 at 
  ph 8 ( one - way analysis of variance test ) . within the same condition ,
ros rates 
  taken at each ph were significantly different from that at the adjacent ph 
  value ( n = 310 , p < 0.05 ) , with the exceptions of 
  the rates taken at ph 7.5 and 8 in condition 3 ( t test ) .
b , 
  the levels of membrane potential measured at ph 7.08.0 for condition 1 
  were significantly different from that in condition 2 or 3 ( n = 
  38 ; , p < 0.05 , analysis of variance test ) and 
  also from the membrane potential at ph 6.06.5 for the same condition 
  ( n = 3 ; + , p < 0.05 , t test ) .
c , at ph 
  8.0 , rates of respiration measured in conditions 2 and 3 were significantly 
  different from that in condition 1 ( n = 38 ; + , p < 
  0.05 , analysis of variance test ) . in condition 1 , rates at ph 8.0 were 
  significantly different from that at all other ph values ( n = 
  78 ; ,
p < 0.0001 , t test ) ; in 
  condition 3 , rates at ph 7.5 were significantly different from that at ph 6.0 
  and 6.5 ( n = 34 ; # , p < 0.05 , t 
  test ) . in the experiments described above , succinate was applied as a substrate . 
  used in much higher concentrations than normally produced in krebs cycle
, it 
  stimulates high ros production rate , and this was convenient to study the 
  process , although the ros production rates were much higher than could be 
  observed under physiological conditions .
this high concentration of succinate 
  could be observed , however , at pathologies like ischemia 
  ( 27 , 
  28 ) .
more physiological 
  substrates , glutamate and malate , produce much smaller amounts of ros 
  ( 11 , 
  29 ) . however , as 
  fig .
7a illustrates , 
  the rates of ros release in this case also significantly increased with the 
  increase of medium ph .
these experiments were performed in the presence of 
  both pi and nigericin to ensure the conversion of the ph 
  component of proton motive force into the electric one ( condition 3 ) . 
 
fig . 7 , b and 
  c , shows that , indeed , the addition of inorganic 
  phosphate and nigericin did increase membrane potential the same way as it was 
  observed with succinate as substrate .
figure 4.spontaneous depolarization of mitochondria takes place in the medium 
  without pi ( condition 1 ) at ph 8 . a , membrane 
  potential
2 m csa , 
  200 m adp , and 2 m oligomycin were present in 
  the medium before the addition of mitochondria ( curve 2 ) .
spontaneous depolarization of mitochondria takes place in the medium 
  without pi ( condition 1 ) at ph 8 . a , membrane 
  potential ; b , ros .
2 m csa , 
  200 m adp , and 2 m oligomycin were present in 
  the medium before the addition of mitochondria ( curve 2 ) .
7b ) 
  and dramatically stimulated ros generation 
  ( 11 , 
  29 ) . 
 
8 shows that upon 
  application of rotenone , regardless of depolarization , the rate of ros 
  generation increased with the increase of medium ph .
thus , all presented data 
  show the same pattern of ros production increase in conjunction with the 
  alkalization of medium .
in a study performed recently by lambert and brand 
  ( 30 ) , the authors came to the 
  conclusion that ph gradient through the inner mitochondrial membrane 
  determines ros production .
our results clearly show that even in the absence 
  of ph gradient ( figs . 
 
3a ( circles ) , 
  7a , and 
  8) medium ph is an essential 
  factor defining ros production by the respiratory chain , and ph increase 
  itself induces the increase in ros generation these data are in agreement with 
  an early study presented by turrens and boveris 
  ( 31 ) , where a simpler model of 
  submitochondrial particles was used .
the authors have shown that the rate of 
  superoxide generation increased in conjunction with an increase of medium ph 
  from 7 to 9.2 in the presence of nadh or succinate as substrates and 
  inhibitors rotenone or antimycin a , respectively .
figure 5.inorganic phosphate ( a ) and nigericin ( b ) increase the 
  value of membrane potential but decrease the rate of ros release . the same 
  relative scale of fluorescence was used for both dyes .
a , membrane 
  potential ( traces 13 ) and ros ( traces 46 ) . 
 
basic incubation medium , ph 7 , was used ( see  experimental 
  procedures  ) ; 1 mm pi was present ( traces 
  1 and 4 ) , or no pi was present ( traces 2 , 
  3 , 5 , and 6 ) .
the additions of mitochondria 
  ( mt ) ( all traces ) , 1 mm pi ( traces 
  3 and 6 ) and 200 nm fccp ( traces 13 ) 
  were made at the time points indicated by arrows .
b , basic 
  incubation medium without pi was used ; ph was adjusted to 6.5 . 
 
trace 1 , ros ; trace 2 , membrane potential . at the time 
  points indicated by arrows ,
the following additions were made : 
  mitochondria ( all traces ) , 100 nm nigericin ( all 
  traces ) , and 200 nm fccp ( trace 2 ) .
inorganic phosphate ( a ) and nigericin ( b ) increase the 
  value of membrane potential but decrease the rate of ros release .
a , membrane 
  potential ( traces 13 ) and ros ( traces 46 ) . 
 
basic incubation medium , ph 7 , was used ( see  experimental 
  procedures  ) ; 1 mm pi was present ( traces 
  1 and 4 ) , or no pi was present ( traces 2 , 
  3 , 5 , and 6 ) .
the additions of mitochondria 
  ( mt ) ( all traces ) , 1 mm pi ( traces 
  3 and 6 ) and 200 nm fccp ( traces 13 ) 
  were made at the time points indicated by arrows .
b , basic 
  incubation medium without pi was used ; ph was adjusted to 6.5 . 
 
trace 1 , ros ; trace 2 , membrane potential . at the time 
  points indicated by arrows ,
the following additions were made : 
  mitochondria ( all traces ) , 100 nm nigericin ( all 
  traces ) , and 200 nm fccp ( trace 2 ) .
the ph dependence of ros production could be explained mechanistically , as 
  outlined in the introduction .
proton binding could be a limiting step in 
  semiquinone radical - ubiquinol transformation ; in this case , the decrease of 
  proton concentration must stabilize semiquinone radical .
ohnishi and trumpower 
  ( 32 ) directly measured that 
  semiquinone radical concentration in respiratory chain steeply increased with 
  the increase of ph .
this supports the proposed role of medium ph in 
  mitochondrial ros generation by respiratory chain .
figure 6.ionophores valinomycin and fccp have opposite effect on the rate of ros 
  release by brain mitochondria . a , recordings of membrane 
  potential
basic medium without 
  pi ( ph 6 ) was used . at the time points indicated by 
  arrows ,
the following additions were made : mitochondria ( mt ) 
  ( all traces ) , 20 pm valinomycin ( traces 1a and 
  1b ) , and 2.5 nm fccp ( traces 2a and 2b ) . 
 
concentration of k ions in the medium was 125 mm . to 
  achieve a complete depolarization ,
ionophores valinomycin and fccp have opposite effect on the rate of ros 
  release by brain mitochondria . a , recordings of membrane 
  potential
basic medium without 
  pi ( ph 6 ) was used . at the time points indicated by 
  arrows ,
the following additions were made : mitochondria ( mt ) 
  ( all traces ) , 20 pm valinomycin ( traces 1a and 
  1b ) , and 2.5 nm fccp ( traces 2a and 2b ) . 
 
concentration of k ions in the medium was 125 mm . to 
  achieve a complete depolarization ,
the experimental results of 
  ( 30 ) are in line with the ones 
  presented here , and we believe that they could be better interpreted in terms 
  of absolute value of matrix ph .
indeed , ph = phin - 
  phout could be increased by two ways ( either by increasing 
  phin or by decreasing phout ) with a different outcome . 
 
higher phin would stabilize semiquinone radical on the matrix side 
  of the membrane , thus stimulating ros production , and this was observed in the 
  work ( 30 ) as well as in the 
  present study .
however , lowering outside ph ( increase of outside proton 
  concentration ) would counteract the dissociation of protons from ubiquinol and 
  formation of semiquinone radical ( reaction 
  2 ) . in the latter case
3a indicates that in the same medium ph , ros production 
  is different , apparently because the matrix ph is different as defined by the 
  respective conditions of incubation .
the decrease of matrix ph induced by the 
  addition of pi and nigericin ( switch from condition 1 to condition 
  2 or 3 ) is accompanied by an increase of  
  ( fig .
3b ) , which is 
  expected to stimulate ros production . however , the ros generation rate 
  decreased ( figs .
3a 
  ( triangles and circles ) and 
  5 ) , thus indicating that in 
  these circumstances the effect of ph dominates over the opposite effect of 
  . figure 7.effect of medium ph on rate of ros release ( a ) and level of 
  membrane potential of mitochondria oxidizing complex i substrates glutamate 
  and malate ( b and c ) .
basic incubation medium was 
  supplemented with 1 mm pi ( a and c ) 
  and 100 nm nigericin ( a ) .
for traces of membrane 
  potential ( b and c ) , ph was adjusted to the values indicated 
  by each trace .
the additions of mitochondria ( mt ) , 1 mm 
  pi , 1 m rotenone , 100 nm nigericin , and 
  200 nm fccp were made at the time points indicated by the 
  arrows .
all data sets for ros 
  rates were statistically different from each other ( n = 4 ; 
  , p < 0.05 , t test ) .
effect of medium ph on rate of ros release ( a ) and level of 
  membrane potential of mitochondria oxidizing complex i substrates glutamate 
  and malate ( b and c ) .
basic incubation medium was 
  supplemented with 1 mm pi ( a and c ) 
  and 100 nm nigericin ( a ) . for traces of membrane 
  potential ( b and c ) ,
the additions of mitochondria ( mt ) , 1 mm 
  pi , 1 m rotenone , 100 nm nigericin , and 
  200 nm fccp were made at the time points indicated by the 
  arrows .
all data sets for ros 
  rates were statistically different from each other ( n = 4 ; 
  , p < 0.05 , t test ) .
the mentioned above comparison of the three conditions could give an 
  approximate quantitative estimation of ph gradient and the role of matrix ph 
  in ros production .
in the presence of nigericin , matrix ph is the same as 
  external ph ; therefore , subtracting the ros production rate under condition 3 
  from that under two other conditions ( as indicated in 
  fig .
9 ) , we can deduce the role 
  of matrix ph difference in ros production . as 
  fig . 9 shows , at ph 6 , the 
  difference between ros production rate under the condition when pi 
  and nigericin are absent ( condition 1 ) and when they are present ( condition 3 ) 
  is 650 pmol / min / mg protein .
the reason for this difference is different matrix 
  ph , which is higher in the absence of pi and nigericin ( condition 
  1 ) .
the same difference in ros production ( 650 pmol / min / mg prot ) could be 
  induced by a 0.5-unit ph shift from ph 6 to 6.5 in the presence of both 
  pi and nigericin ( under condition 3 ) 
  ( fig .
therefore , if under condition 1 matrix ph defines this 
  difference in ros production , it must be not less than 6.5 when ph of the 
  medium is 6 .
figure 8.acidic ph inhibits rotenone - induced ros generation by rbm in the 
  presence of glutamate and malate .
basic incubation medium supplemented 
  with 1 mm pi was used ; concentration of rotenone was 1 
  m .
data sets taken at ph 
  6.0 , 6.5 , and 7.0 were significantly different from that at ph 7.5 and 8.0 
  ( n = 58 ; , p < 0.01 ; , 
  p < 0.001 , t test ) .
acidic ph inhibits rotenone - induced ros generation by rbm in the 
  presence of glutamate and malate .
basic incubation medium supplemented 
  with 1 mm pi was used ; concentration of rotenone was 1 
  m .
data sets taken at ph 
  6.0 , 6.5 , and 7.0 were significantly different from that at ph 7.5 and 8.0 
  ( n = 58 ; , p < 0.01 ; , 
  p < 0.001 , t test ) .
figure 9.difference between the rates of ros release calculated from the data 
  presented in fig . 
  3 .
results of subtraction of ros release rates are 
  plotted versus corresponding ph of the medium .
circles , 
  condition 1 to condition 3 ; squares , condition 2 to condition 3 . 
 
values of difference between condition 1 
  and condition 3 ( circles ) at ph 6.5 and 7 were significantly 
  different from that at ph 6 ( n = 310 ; , 
  p
< 0.01 , t test ) ; values of difference between condition 
  2 and condition 3 ( squares ) at ph 7 were significantly different from 
  that at ph 8 ( n = 310 ; , p < 0.05 , 
  t test ) .
difference between the rates of ros release calculated from the data 
  presented in fig . 
 
results of subtraction of ros release rates are 
  plotted versus corresponding ph of the medium .
circles , 
  condition 1 to condition 3 ; squares , condition 2 to condition 3 . 
 
values of difference between condition 1 
  and condition 3 ( circles ) at ph 6.5 and 7 were significantly 
  different from that at ph 6 ( n = 310 ; , 
  p
< 0.01 , t test ) ; values of difference between condition 
  2 and condition 3 ( squares ) at ph 7 were significantly different from 
  that at ph 8
( n = 310 ; , p < 0.05 , 
  t test ) . at ph 7 ,
the difference between ros production under conditions 1 and 3 is 
  even higher ( 1300 pmol / min / mg protein ( fig . 
  9 , circles ) ) .
using the same reasoning as above , we can 
  deduce that under condition 1 at ph 7 , an even higher difference between 
  external and internal ph should be expected .
thus , high ph must result 
  in  decrease , and it was measured as 
  fig .
moreover , so high a ph difference assumes extremely 
  low intramitochondrial proton concentrations ; this could be a limiting factor 
  restricting respiration rate when mitochondria are in state 3 , as demonstrated 
  by fig .
this indirect estimation of ph gradient by ros production is in agreement with 
  measurements made by conventional methods , 0.51.4 ph units 
  ( 30 , 
  33 , 
  34 ) .
redox reactions in electron transport chain and proton 
  translocation are fed by glycolysis and the krebs cycle , which provide nadh , 
  complex i substrate , and succinate , complex ii substrate .
respiratory complex 
  i accepts electrons from nadh , oxidizing it to nad , and delivers 
  these electrons to ubiquinone ( q ) .
proton transport in complex iii is coupled 
  with electron transport in accordance with the generally accepted 
  ubiquinone / ubiquinol ( q / qh2 ) cycle mechanism , which is shown in 
  more detail .
the overall reaction performed by complex iii is as follows , 
  qh2 + 2cyt cox + 2hn  q + 2cyt 
  cred + 4hp(i.e .
it oxidizes ubiquinol , 
  reducing cytochrome c and releasing two h to the 
  cytosolic side ( positive or p - side ; this is reflected in the index of released 
  h ) ) .
in addition , it translocates two protons from matrix 
  ( negative or n - side ) to cytosol .
ubiquinol ( qh2 ) delivers its first 
  electron to fe , releasing two protons at the p - side of the inner 
  mitochondrial membrane and producing semiquinone radical ( sq ) . 
 
then the latter gives its unpaired electron to cytochrome 
  bl , and produced ubiquinone ( q ) dissociates from the 
  complex .
free q binds at the n - side and receives two electrons from cytochrome 
  bh , resulting from oxidation of two qh2 
  molecules , thus producing subsequently sq and qh2 , 
  taking protons from the n - side .
redox reactions in electron transport chain and proton 
  translocation are fed by glycolysis and the krebs cycle , which provide nadh , 
  complex i substrate , and succinate , complex ii substrate .
respiratory complex 
  i accepts electrons from nadh , oxidizing it to nad , and delivers 
  these electrons to ubiquinone ( q ) .
proton transport in complex iii is coupled 
  with electron transport in accordance with the generally accepted 
  ubiquinone / ubiquinol ( q / qh2 ) cycle mechanism , which is shown in 
  more detail .
the overall reaction performed by complex iii is as follows , 
  qh2 + 2cyt cox + 2hn  q + 2cyt 
  cred + 4hp(i.e .
it oxidizes ubiquinol , 
  reducing cytochrome c and releasing two h to the 
  cytosolic side ( positive or p - side ; this is reflected in the index of released 
  h ) ) .
in addition , it translocates two protons from matrix 
  ( negative or n - side ) to cytosol .
ubiquinol ( qh2 ) delivers its first 
  electron to fe , releasing two protons at the p - side of the inner 
  mitochondrial membrane and producing semiquinone radical ( sq ) . 
 
then the latter gives its unpaired electron to cytochrome 
  bl , and produced ubiquinone ( q ) dissociates from the 
  complex .
free q binds at the n - side and receives two electrons from cytochrome 
  bh , resulting from oxidation of two qh2 
  molecules , thus producing subsequently sq and qh2 , 
  taking protons from the n - side .
ros levels are physiologically important as a metabolic signal 
  ( 35 , 
  36 ) .
ros overproduction is one 
  of the main factors inducing apoptosis 
  ( 37 ) .
here we show that 
  mitochondrial matrix ph is one of the factors controlling ros signaling .
higher matrix ph in the absence of pi and nigericin ( condition 
  1 ) at medium ph more than 7 resulted in spontaneous depolarization , probably 
  due to permeability transition as figs . 
 
3b ( squares ) 
  and 4a illustrate . in 
  these conditions , mitochondria experience dramatic perturbations with the loss 
  of all gradients across the inner membrane .
this resulted in a substantial 
  decrease of ros release , which requires functional integrity of the 
  mitochondrial membrane ( figs . 
 
10 that 
  sq is not produced if the electron efflux from the fes center is 
  blocked . on the other hand ,
matrix ph was shown to be an essential factor in 
  induction of permeability transition 
  ( 38 ) .
evidence from the 
  literature indicates that the induction of permeability transition may occur 
  in the presence of inorganic phosphate and/or ca 
  ( 39 ) . since these compounds 
  were not present in the medium of condition 1 ,
our results demonstrate that ph 
  itself could regulate permeability transition ; this proves the conclusion of 
  our previous theoretical study 
  ( 40 ) .
the permeability transition was not observed under conditions 2 and 3 ; 
  however , the alkalization - induced increase of respiration rate when 
   remained constant ( figs . 
 
3c , triangles 
  and circles ) indicates that ros affects membrane permeability , 
  increasing leaks . in the presence of pi and nigericin ( under 
  condition 3 ) ,
respiration increased more steeply with ph increase despite 
  slower ros production ( fig . 
 
this could be due to the fact that 
   in the presence of both pi and nigericin ( condition 3 ) 
  is higher and that the leak strongly depends on . at ph 8 , 
  respiration decreases (  remains unchanged ) , apparently due to the 
  decrease in proton leak , since external h concentration decreases . 
 
steep inhibition of respiration observed in condition 1 at ph 7.5 and 8 , in 
  all likelihood , is the consequence of the loss of cytochrome c in the 
  course of permeability transition .
the data discussed above leave no doubts that change in matrix ph is vital ; 
  it controls such physiologically important processes as ros production and 
  permeability transition .
proton translocation coupled with 
  electron transport itself can not change ph extensively , because a small amount 
  of translocated protons results in high increase of electric potential , which 
  stops the process . however , if proton translocation is coupled with an influx 
  of some cations , such as ca or k , it could result in 
  a considerable alkalization of matrix .
mitochondria are also permeable for ca , and it is 
  possible that ca overload of cells and , consequently , 
  mitochondria could take place under some stress conditions .
although proton 
  leak normally is compensated by respiratory proton translocation , it could set 
  a dynamic steady state with matrix less alkaline .
application of uncoupler 
  fccp shown in fig . 6 modulates 
  this situation . in cells ,
the activation of ucps can down - regulate the level of ros by controlling the 
  current of protons into the matrix without considerable compromise of 
  mitochondrial atp production . to demonstrate the phenomenon of ph regulation of ros production by the 
  respiratory chain
, mitochondria were studied in the conditions of artificially 
  high succinate levels . in this case
, they produce ros with a higher rate than 
  when respiring on physiological substrates glutamate and malate ( although with 
  similar ph dependence , as figs . 
 
ph , 
  evidently , is not the only factor defining ros production rate . if the levels 
  of ubiquinol are high and , according to mass conservation , the levels of 
  ubiquinone are low , the lack of electron acceptor ubiquinone results in a 
  block of electron efflux from cytochrome bh and , 
  subsequently , bl ( see 
  fig .
the levels of cytosolic side semiquinone radical and , consequently , the rate 
  of ros production must be high .
oxidation of succinate , available in excess , 
  produces much higher levels of ubiquinol than would be produced by complex i , 
  since ubiquinone reduction by respiratory complex ii is not related with 
  proton translocation ( as in complex i ) and is not restricted by the proton 
  electrochemical gradient .
we suggest that this is the main reason why 
  mitochondria , when respiring on succinate , produce much more ros than when 
  they respire on complex i substrates .
it should be noted that the increase of matrix ph stabilizes not only 
  matrix side but also cytosolic side semiquinone .
the stabilization of matrix 
  side semiquinone slows down the electron transport in the chain upstream , thus 
  increasing the life - time of semiquinone radical bound to cytosolic side .
this 
  rationale is in accordance with the finding that complex iii of mitochondria 
  respiring on succinate produces substantial amounts of ros on the cytosolic 
  side of the membrane ( 42 , 
  43 ) .
the whole set of experiments presented here supports the proposed 
  ph - dependent mechanism of ros generation in mitochondrial respiratory chain 
  and its link with mitochondrial permeability transition .
it allows us to 
  understand the mechanism by which ucps down - regulate the level of ros in 
  cells .
the specific properties and quantitative data featuring mitochondrial 
  ros generation could be a basis for a more detailed study aimed at exploration 
  of the mechanism of ros production .
 reactive oxygen species ( ros ) generation in mitochondria as a side product 
  of electron and proton transport through the inner membrane is important for 
  normal cell operation as well as development of pathology . matrix and cytosol 
  alkalization stabilizes semiquinone radical , a potential superoxide producer , 
  and we hypothesized that proton deficiency under the excess of electron donors 
  enhances reactive oxygen species generation . 
 we tested this hypothesis by 
  measuring ph dependence of reactive oxygen species released by mitochondria . 
  
 the experiments were performed in the media with ph varying from 6 to 8 in the 
  presence of complex ii substrate succinate or under more physiological 
  conditions with complex i substrates glutamate and malate . 
 matrix ph was 
  manipulated by inorganic phosphate , nigericine , and low concentrations of 
  uncoupler or valinomycin . 
 we found that high ph strongly increased the rate of 
  free radical generation in all of the conditions studied , even when ph 
  = 0 in the presence of nigericin . in the absence of inorganic phosphate , when 
  the matrix was the most alkaline 
 , ph shift in the medium above 7 induced 
  permeability transition accompanied by the decrease of ros production . 
 ros 
  production increase induced by the alkalization of medium was observed with 
  intact respiring mitochondria as well as in the presence of complex i 
  inhibitor rotenone , which enhanced reactive oxygen species release . 
 the 
  phenomena revealed in this report are important for understanding mechanisms 
  governing mitochondrial production of reactive oxygen species , in particular 
  that related with uncoupling proteins . 
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION