4.1. Complex contribution of cyclophilin-D in brain-specific mitochondrial permeability transition induced by Ca
2 +4.1.1. Effect of substrate availability on Ca2 +-induced PTP and modulation by cyclosporin A or genetic deletion of cyclophilin-D
Electrophoretic Ca2 + uptake for induction of PTP is allowed either in the presence of respiratory substrates or in a substrate-free medium containing KSCN; diffusion of the lipophilic SCN- anion provides the driving force for electrophoretic Ca2 + accumulation [174;175]. However, in the original studies by Hunter and Haworth it was shown that PTP can be induced by Ca2 + in the absence of respiratory substrates [73;73], a phenomenon that has been subsequently reproduced [176-178], reviewed in [179]. Furthermore, it was also shown that substrates delay Ca2 + -induced PTP [176;177], in accordance to the Bernardi scheme elaborated above. To reproduce these findings for our studies, isolated brain mitochondria were challenged by CaCl2, in the presence and absence of glutamate and malate, and light scattering was recorded spectrophotometrically at 520 nm. A three-pulse CaCl2 protocol was used for this, and all subsequent similar experiments: 20 µ M CaCl2 was given at 100 sec, followed by 200 µ M CaCl2 at 300 sec and again at 500 sec.
Orange lines appearing in Figures 5, 6, and 8 represent traces obtained from WT mitochondria incubated in such conditions that reproduced the most pronounced swelling induced by CaC2 (respective for each panel), but these mitochondria were not challenged by CaCl2.
Complex contribution of Cyclophilin-D in brain-specific mitochondrial permeability transition induced by Ca2+
F i g u r e 5 . E f f e c t o f s u b s t r a t e a v a i l a b i l i t y o n C a2 +- i n d u c e d P T P a n d m o d u l a t i o n b y c y c l o s p o r i n A o r g e n e t i c d e l e t i o n o f c y c l o p h i l i n - D . A , B , D , E : T r a c e s o f l i g h t s c a t t e r r e c o r d e d s p e c t r o p h o t o m e t r i c a l l y i n m i t o c h o n d r i a l s u s p e n s i o n s a t 5 2 0 n m d u r i n g C a C l2 a d d i t i o n s a t t h e c o n c e n t r a t i o n s i n d i c a t e d i n t h e f i g u r e s . C o n d i t i o n s a r e g i v e n i n t h e p a n e l s . C : W e s t e r n b l o t ( u p p e r p a n e l ) o f b r a i n a n d l i v e r m i t o c h o n d r i a f r o m W T v s K O C y p D m i c e p r o b e d f o r C y p D i m m u n o r e a c t i v i t y . L o w e r p a n e l : P o n c e a u S s t a i n i n g o f t h e s a m e b l o t s h o w n i n t h e u p p e r p a n e l . M W M : M o l e c u l a r W e i g h t M a r k e r . P a n e l s D a n d E a r e a l i g n e d o n t h e y - a x i s . P a n e l s A , D , E a n d F a r e a l i g n e d o n t h e x - a x i s . F : M i t o c h o n d r i a l C a2 + u p t a k e f o l l o w e d b y C a l c i u m G r e e n 5 N h e x a p o t a s s i u m s a l t f l u o r e s c e n c e ( n o n - c a l i b r a t e d ) . T h e b l a c k s t r i p p e d r e g i o n i s e x p a n d e d o n t h e y - a x i s , f o r t h e s a k e o f c l a r i t y ; a : W T , p l u s g l u t a m a t e p l u s m a l a t e ; b : W T , p l u s g l u t a m a t e p l u s m a l a t e p l u s C y s A ; c : W T , n o s u b s t r a t e s ; d : W T , n o s u b s t r a t e s p l u s C y s A ; e : W T , n o s u b s t r a t e s p l u s R u 3 6 0 . R e s u l t s a r e r e p r e s e n t a t i v e o f a t l e a s t 4 i n d e p e n d e n t e x p e r i m e n t s . P a n e l G d e p i c t s t h e m a x i m u m s w e l l i n g r a t e s p o o l e d f r o m a l l i n d i v i d u a l e x p e r i m e n t s ( e x p r e s s e d a s p e r c e n t a g e o f s w e l l i n g r a t e p e r m i n u t e a n d
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As shown in Figure 5 A, addition of 20 µ M CaCl2 to substrate-supplemented or substrate-starved brain mitochondria of wild type mice did not cause a decrease in light scatter; instead, a cessation in the baseline decrease in light scatter was observed. However, the subsequent 200 µ M CaCl2 pulse induced a large decrease in light scatter in substrate-starved but not substrate-supplemented mitochondria. The next 200 µ M CaCl2 pulse given at 500 sec did not induce any further changes in light scatter for the substrate-starved mitochondria, but it caused a minor change in substrate-supplemented mitochondria. As shown in Fig.
5, panel B, the effect of the first 200 µ M CaCl2 pulse was Cyclosporin changes. Subsequent experiments benefited from the availability of cyclophilin-D knock-out mice. We isolated mitochondria from the brains of WT and CypD-KO mice (see Figure 5, panel C). As shown in panel D, results obtained from substrate-starved mitochondria from CypD-KO mice were strikingly similar to those obtained from CysA-treated WT mice (panel B). The presence of substrates, however, did not provide additional protection in the CypD-KO mitochondria (panel E). Maximum swelling rates pooled from all experiments (expressed as percentage of swelling rate per minute and accounting for the condition producing the highest swelling rate as ‘maximum’) for each condition and after each Ca2 + pulse is shown in Fig. 5, panel G. These results are in accord to earlier reports on various types of mitochondria and conditions, showing that high-Ca2 + loads can induce PTP in the absence of substrates. In our hands, absence of substrates prevented isolated mitochondria from building a membrane potential of higher than -10 mV (not shown). At this ∆Ψm value, mitochondrial Ca2 + uptake is unfavorable [51]. Indeed, recordings of extramitochondrial Ca2 + by Calcium Geeen 5N revealed
Complex contribution of Cyclophilin-D in brain-specific mitochondrial permeability transition induced by Ca2+
that in the absence of substrates (Figure 5, panel F, traces c, d, and e) mitochondria were unable to perform Ca2 + sequestration, yet exhibited large changes in light scatter. Electron microscopy imaging of mitochondria that exhibited large changes in light scatter confirmed that this was due to swelling (not shown). We therefore considered the possibilities that Ca2 + was either inducing CypD-sensitive swelling by acting on an extramitochondrial site, or since high amounts of CaCl2 were required, Ca2 + was entering mitochondria simply by a chemical gradient.
4.1.2. Effect of an uncoupler and/or inhibitors of the Ca2 + uniporter on mitochondrial Ca2 + uptake and light scatter
To address the site of action of Ca2 + on the light scatter, we pretreated mitochondria with the Ca2 + uniporter inhibitor, Ru360 [21].
As shown in Figure 6 A, WT mitochondria still exhibited high-Ca2 + -induced changes in light scatter in the presence of Ru360, at a concentration that was found to prevent the uptake of extramitochondrial Ca2 + (Figure 5, panel F, trace e). The lack of effect of Ru360 was also swelling. Furthermore, the presence of the uncoupler negated the protective effects of substrates in WT mitochondria (panel D). Maximum swelling rates pooled from all experiments (expressed as in Figure 5) for each condition and after each Ca2 + pulse is shown in panel E of Figure 6.
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F i g u r e 6 . E f f e c t o f S F 6 8 4 7 a n d / o r i n h i b i t o r s o f t h e C a2 + u n i p o r t e r o n m i t o c h o n d r i a l C a2 + u p t a k e ; m o d u l a t i o n b y C y c l o s p o r i n - A o r g e n e t i c d e l e t i o n o f C y c l o p h i l i n - D . T r a c e s o f l i g h t s c a t t e r r e c o r d e d s p e c t r o p h o t o m e t r i c a l l y a t 5 2 0 n m d u r i n g C a C l2 a d d i t i o n s a t t h e c o n c e n t r a t i o n s i n d i c a t e d i n t h e f i g u r e s , t o m i t o c h o n d r i a l s u s p e n s i o n s . C o n d i t i o n s o f t h e s u s p e n s i o n s a r e g i v e n i n t h e p a n e l s . A l l p a n e l s a r e a l i g n e d o n t h e x -a x i s . R e s u l t s -a r e r e p r e s e n t -a t i v e o f -a t l e -a s t 4 i n d e p e n d e n t e x p e r i m e n t s . P -a n e l E d e p i c t s t h e m a x i m u m s w e l l i n g r a t e s p o o l e d f r o m a l l i n d i v i d u a l e x p e r i m e n t s ( e x p r e s s e d a s i n F i g u r e 5 ) f o r e a c h c o n d i t i o n a n d a f t e r e a c h C a2 + p u l s e . E r r o r b a r s r e p r e s e n t S . E . M . ; a i s
Complex contribution of Cyclophilin-D in brain-specific mitochondrial permeability transition induced by Ca2+
4.1.3. Effect of Ca2 + uniporter inhibitors on mitochondrial matrix Ca2 + accumulation of isolated mitochondria imaged under wide-field epifluorescence
The failure of Ru360 to protect against the Ca2 +-induced large changes in light scatter shown in Figures 6 A and 6 B could be explained by assuming that Ca2 + acted on the extramitochondrial side. To
F i g u r e 7 . E f f e c t o f t h e C a2 + u n i p o r t e r i n h i b i t o r s R u 3 6 0 a n d R u t h e n i u m R e d o n m i t o c h o n d r i a l m a t r i x C a2 + a c c u m u l a t i o n o f i s o l a t e d W T b r a i n m i t o c h o n d r i a i m a g e d u n d e r w i d e - f i e l d e p i f l u o r e s c e n c e . A : W i d e - f i e l d e p i f l u o r e s c e n c e i m a g e o f i s o l a t e d b r a i n m i t o c h o n d r i a f r o m W T m i c e , l o a d e d w i t h f u r a 2 A M . B : T i m e r e c o r d i n g s o f m i t o c h o n d r i a l - t r a p p e d f u r a 2 f l u o r e s c e n c e o f i m m o b i l i z e d m i t o c h o n d r i a , p e r f u s e d b y 0 . 1 m M C a C l2 i n t h e p r e s e n c e o f a b s e n c e o f C a2 + u n i p o r t e r i n h i b i t o r s a s i n d i c a t e d i n t h e p a n e l , i n t h e a b s e n c e o f e x o g e n o u s s u b s t r a t e s . R e s u l t s a r e r e p r e s e n t a t i v e o f a t l e a s t 4
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provide further evidence for this, we loaded isolated mitochondria with Fura 2, and imaged them under wide-field epifluorescence (Figure 7, panel A). This experimental setup benefits from i) the spatial resolution in Fura 2 imaging, avoiding a “contaminant” signal of leaked Fura 2 in the extramitochondrial space and ii) provides a valid quantitative signal of matrix [Ca2 +] in the submicromolar range. Surprisingly, isolated mitochondria perfused with a buffer containing 0.1 mM CaCl2 showed robust increases in matrix-entrapped Fura 2 fluorescence that exhibited only a partial sensitivity to Ru360 (10 µ M) and ruthenium red (10 µ M), panel B, arguing against the assumption that Ca2 + was acting exclusively on an extramitochondrial site when inducing changes in light scatter.
4.1.4. Effect of respiratory chain inhibition on Ca2 +-induced PTP
To address the contribution of respiratory chain components to the protective effect of substrates on Ca2 +-induced changes in light scatter, we pretreated mitochondria with complex I (rotenone or piericidin A), complex III (myxothiazol or stigmatellin), and complex IV (KCN) inhibitors. The emerging picture depicted from Figure 8 was that rotenone or piericidin A (traces c, d of panel A and traces b, c of panel B) afforded protection from Ca2 +-induced changes in light scatter, irrespective of the presence or abscence of substrates, while myxothiazol, stigmatellin and KCN not only falied to confer protection, they also negated the protective effect of substrates (traces e, f of panel A and traces b, c, d, e, f of panel C of Fig. 8; see also Figure 8, panel D). High concentrations of myxothiazol and stigmatellin (10 µ M and 2 µ M, respectively) that also block complex I [181], failed to afford protection, as opposed to rotenone and piericidin A. However, rotenone and piericidin A (as well as rolliniastatin used in [80]), bind to a different site than myxothiazol and stigmatellin [181]. Maximum swelling rates pooled from all experiments (expressed as in Figures 5 and 6) for each condition and after each Ca2 + pulse is shown in panel D
Complex contribution of Cyclophilin-D in brain-specific mitochondrial permeability transition induced by Ca2+
of Figure 8. Inhibition of PTP by rotenone has been previously reported
F i g u r e 8 . E f f e c t o f r e s p i r a t o r y c h a i n i n h i b i t i o n o n C a2 +- i n d u c e d P T P . T r a c e s o f l i g h t s c a t t e r r e c o r d e d s p e c t r o p h o t o m e t r i c a l l y a t 5 2 0 n m d u r i n g C a C l2 a d d i t i o n s a t t h e c o n c e n t r a t i o n s i n d i c a t e d i n t h e f i g u r e s , t o m i t o c h o n d r i a l s u s p e n s i o n s . A l l e x p e r i m e n t s w e r e p e r f o r m e d o n W T m i c e . A : a : n o s u b s t r a t e s ; b : p l u s g l u + m a l ; c : p l u s g l u + m a l + 1 µ M R o t e n o n e ; d : n o s u b s t r a t e s + 1 µ M R o t e n o n e ; e : n o s u b s t r a t e s + 1 m M K C N ; f : p l u s g l u + m a l + 1 m M K C N . B : a : n o s u b s t r a t e s ; b : n o s u b s t r a t e s + 1 µ M P i e r i c i d i n A ; c : n o s u b s t r a t e s + 1 µ M R o t e n o n e . C : a : n o s u b s t r a t e s ; b : n o s u b s t r a t e s + 0 . 2 µ M s t i g m a t e l l i n ; c : n o s u b s t r a t e s + 2 µ M s t i g m a t e l l i n ; d : n o s u b s t r a t e s + 0 . 5 µ M m y x o t h i a z o l ; e : n o s u b s t r a t e s + 1 0 µ M m y x o t h i a z o l ; f : n o s u b s t r a t e s + 1 m M K C N . R e s u l t s a r e r e p r e s e n t a t i v e o f a t l e a s t 4 i n d e p e n d e n t e x p e r i m e n t s . P a n e l D d e p i c t s t h e m a x i m u m s w e l l i n g r a t e s p o o l e d f r o m a l l i n d i v i d u a l
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[73;80;177;182]. Since the protective effect of substrates was negated by cyanide, we decided to use this regimen for testing mitochondrial swelling within neurons and astrocytes, since the in situ availability of substrates is much less amenable to manipulation.
On Table 2, the effect of complex I, III and IV inhibitors on Ca2 + -induced PTP depicted in Figure 8 is summarized, highlighting the fact that only Rotenone and Piericidin A binding to complex I on a specific binding site are protective, while inhibition of the respiratory chain elsewhere even negates the protective effect of substrates.
Table 2. E f f e c t o f r e s p i r a t o r y c h a i n i n h i b i t o r s o n C a2 +- i n d u c e d P T P .
Complex contribution of Cyclophilin-D in brain-specific mitochondrial permeability transition induced by Ca2+
4.1.5 Effect of CypD ablation on Ca2 +-induced mitochondrial swelling within neurons and astrocytes
To address the role of CypD in the opening of brain-specific PTP in situ, swelling of CypD-deficient versus wild type mitochondria within neurons or astrocytes of the same culture were compared during Ca2 + overload, induced by addition of calcimycin (1 µ M 4Br-A23187).
Mitochondria were visualized by wide field epifluorescence imaging of mitochondrially targeted DsRed2. Astrocytes grew as a monolayer on the bottom of the chamber, while neurons grew as a monolayer above them.
Cultures were prepared at the same age from the WT and KO mouse pups (P0 or 1) and measurements were performed in the same range of days in vitro; accordingly, the neurons/astrocytes ratio deviated minimally from one culture to another (2-2.6), deduced from counting cells from images of fura-loaded cultures. Neurons and astrocytes were distinguished by their different mitochondrial morphology [183]. Neuronal mitochondria are typically more densely packed in the soma, therefore dendritic mitochondria were chosen for analysis. Astrocytes are flatter than neurons in culture, and they exhibited elongated, branched mitochondria of even thickness. Mitochondrial swelling was monitored by evaluating DsRed2-visualized mitochondrial morphology by calculation of changes in mean mitochondrial diameters using the thinness ratio technique. In these assays the onset of swelling was defined by the sudden decrease in the thinness ratio. As shown in Figure 9 A, both WT and CypD-KO mitochondria within neurons swelled and fragmented in response to Ca2 + overload induced by the addition of calcimycin within 600-800 sec.
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F i g u r e 9 . E f f e c t o f C y p D a b l a t i o n o n C a2 +- i n d u c e d m i t o c h o n d r i a l s w e l l i n g w i t h i n n e u r o n s a n d a s t r o c y t e s . A - B : T i m e e l a p s e d b e t w e e n c a l c i m y c i n a p p l i c a t i o n a n d m i t o c h o n d r i a l s w e l l i n g w a s d e t e c t e d b y w i d e f i e l d i m a g i n g o f m i t o - D s R e d 2 e x p r e s s i n g n e u r o n s ( A ) a n d a s t r o c y t e s ( B ) i n m i x e d c o r t i c a l c u l t u r e s f r o m w i l d t y p e ( b l a c k b a r s ) o r C y p D - K O ( g r a y b a r s ) m i c e . T h e o n s e t o f s w e l l i n g w a s d e t e r m i n e d b y d e t e c t i o n o f a n i n c r e a s e o f m e a n d i a m e t e r o f m i t o c h o n d r i a i n t h e m i c r o s c o p i c v i e w f i e l d b y c a l c u l a t i o n o f t h e t h i n n e s s r a t i o . C o - a p p l i c a t i o n o f 5 m M N a C N a n d c a l c i m y c i n w a s p e r f o r m e d i n a m e d i u m w i t h o u t g l u c o s e s u p p l e m e n t e d w i t h 2 m M 2 - d e o x y g l u c o s e . B a r s i n d i c a t e m e a n s ± S . E . M . o f 4 - 1 2 c e l l s ( * , p < 0 . 0 5 s i g n i f i c a n c e b y K r u s k a l - W a l l i s A N O V A o n R a n k s ) .
Complex contribution of Cyclophilin-D in brain-specific mitochondrial permeability transition induced by Ca2+
When NaCN was co-applied with calcimycin in a glucose-free media in the presence of 2 mM 2-deoxyglucose, swelling of mitochondria was almost immediate in both wild type and CypD-KO neurons.
However, Ca2 + overload of uncoupled mitochondria (by co-application of 1 µ M SF 6847) triggered swelling at a significantly earlier time in CypD-KO than in wild type neurons. In contrast to the neurons, Ca2 + overload of uncoupled mitochondria triggered swelling at a significantly earlier time in wild type than in CypD-KO astrocytes (Figure 9 B). In the absence of glucose and concomitant presence of 2-deoxyglucose and NaCN, in situ mitochondria are almost certainly completely depolarized.
We used calcimycin at 1 µ M concentration that likely affects only the Ca2 +-permeability of the plasma membrane. Experiments on cultured neurons and astrocytes loaded with fura 2 (Kd=225 nM, [184]) versus fura 6F (Kd=2.47 µ M, [170]) revealed that the increase in cytosolic Ca2 + by 1 µ M calcimycin was in the 1-2 µ M range in contrast to the 1.3 mM in the medium (not shown); thus the amount of calcimycin distributed in the plasma membrane was very small and unlikely for it to distribute in the inner mitochondrial membrane.
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4.1.6. Effect of CypD ablation on in situ mitochondrial swelling of neurons challenged by glutamate
In order to expose neuronal in situ mitochondria to high-Ca2 + challenge by an alternative mechanism, neurons were exposed to excitotoxic levels of glutamate and glycine, in the absence of Mg2 +. Mitochondrial swelling was monitored by thinness ratio values calculated from wide field fluorescence images of mitochondrially targeted DsRed2. Glutamate exposure triggered a biphasic mitochondrial swelling response (shown for a WT neuron) indicated by the decrease of the thinness ratio (Figure 10 A, black circles). Swelling comprised of an initial, 1s t and a well separated, delayed 2n d drop. The 1s t drop of thinness ratio invariably coincided with the initial [Ca2 +]i-response to glutamate and the 2n d drop to the secondary, irreversible rise of [Ca2 +]i, termed delayed calcium deregulation, DCD [185-187] (Figure 10 A and B, black bars). In cultures prepared from CypD-KO mice (Figure 10 B, gray bars) the first phase of mitochondrial swelling was detected only in 60% of the neurons. Furthermore, only 40 % of CypD-KO neurons exhibited the secondary swelling of mitochondria during DCD (Fig. 10 B). Finally, the initial swelling of mitochondria was significantly delayed in CypD-KO neurons compared to wild type, while the time of onset of the secondary mitochondrial swelling was not statistically different (Figure 10 C).
Complex contribution of Cyclophilin-D in brain-specific mitochondrial permeability transition induced by Ca2+
F i g u r e 1 0 . E f f e c t o f C y p D a b l a t i o n o n g l u t a m a t e - e v o k e d b i p h a s i c m i t o c h o n d r i a l s w e l l i n g i n c o r t i c a l n e u r o n s . A : W i d e f i e l d f l u o r e s c e n c e t i m e l a p s e r e c o r d i n g i n F u r a -F -F - A M l o a d e d w i l d t y p e c o r t i c a l n e u r o n e x p r e s s i n g m i t o - D s R e d . G l u t a m a t e ( 3 0 0 µ M ) p l u s g l y c i n e ( 1 0 µ M ) i n t h e a b s e n c e o f [ M g2 +]e e v o k e d a r i s e o f [ C a2 +]i i n d i c a t e d b y t h e i n c r e a s i n g r a t i o o f F u r a - F F 3 4 0 / 3 8 0 n m f l u o r e s c e n c e i n t e n s i t i e s ( t r i a n g l e s ) . M i t o c h o n d r i a l s w e l l i n g w a s m e a s u r e d b y c a l c u l a t i n g t h i n n e s s r a t i o ( T R ) o f m i t o - D s R e d f l u o r e s c e n c e i m a g e s ( c i r c l e s ) , w h e r e s w e l l i n g i s m a r k e d b y t h e d e c r e a s i n g t h i n n e s s r a t i o ( a r r o w s ) . T h e 1s t a n d 2n d d r o p s o f t h e t h i n n e s s r a t i o a l w a y s c o i n c i d e d w i t h t h e i n i t i a l r e s p o n s e t o g l u t a m a t e a n d t o t h e D C D , r e s p e c t i v e l y . R e p r e s e n t a t i v e t r a c e s o f 1 0 r e c o r d i n g s .
B : Q u a n t i f i c a t i o n o f t h e o b s e r v a t i o n o f 1s t a n d 2n d d r o p s i n n e u r o n s f r o m w i l d t y p e ( b l a c k b a r s ) a n d C y p D - K O m i c e ( g r e y b a r s ) u s i n g F i s h e r e x a c t t e s t . T o t a l ( n ) c o r r e s p o n d s t o t h e n u m b e r o f c e l l s o b s e r v e d a n d 1s t a n d 2n d d r o p s t o t h e o b s e r v a t i o n o f t h e d r o p s i n t h e t h i n n e s s r a t i o i n r e c o r d i n g s s i m i l a r t o ( A ) . C : T h e o n s e t o f m i t o c h o n d r i a l s w e l l i n g w a s d e f i n e d a s t h e t i m e e l a p s e d b e t w e e n t h e a p p l i c a t i o n o f g l u t a m a t e a n d t h e s u d d e n d e c r e a s e o f t h e t h i n n e s s r a t i o , b a r s s h o w m e a n ± S . E . M . 1 0 c e l l s ( * , p < 0 . 0 5 s i g n i f i c a n c e b y
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4.1.7. Discussion
There are hundreds of publications addressing permeability transition with relation to various aspects of bioenergetics; hereby we attempted to shed light on the phenomenon where CypD-dependent PTP precedes necrotic events during which there is energy crisis, but not apoptosis, a mechanism that is dependent on energy provision. We have narrowed our investigations to brain mitochondria in isolation, and within their natural environment (both neurons and astrocytes).
The most important finding of this study is the dramatic hastening of the swelling of in situ neuronal and astrocytic mitochondria by glucose deprivation and NaCN co-application, upon calcimycin exposure.
This extends the previous findings on isolated mitochondria [81;185;188-191] and the results of this study, showing that a diminished electrochemical gradient primes mitochondria within their natural environment to undergo Ca2 +-induced PTP. We created depolarizing conditions by three different means: i) substrate deprivation (no electron flow), ii) ETC inhibitors (no electron flow), and iii) presence of an uncoupler (high electron flow). Results were not entirely congruent between isolated versus in situ mitochondria (i.e. compare presence versus absence of substrates or uncoupler), but in both experimental models the presence of cyanide prompted pore opening. Our findings support the notion that the lack of an electrochemical gradient unfavoring electrophoretic Ca2 + uptake not only fails to protect in situ mitochondria from PTP, but it also subserves the purpose of decreasing a threshold to the point that either an increase in matrix Ca2 + concentration due to a mere diffusion and/or an extramitochondrial site with low
Complex contribution of Cyclophilin-D in brain-specific mitochondrial permeability transition induced by Ca2+
The swelling induced by high-Ca2 + in energized in situ neuronal and astrocytic mitochondria was not CypD-dependent. To this we must stress that our results on isolated mitochondria point to the possibility of an Ru360/Ruthenium-Red-insensitive route for Ca2 + entry. Furthermore, our results also show that there are cell-specific differences of the same tissue regarding the bioenergetic contribution to Ca2 +-induced PTP. For example, it is striking that the elapsed time upon calcimycin exposure until the onset of swelling of uncoupler-treated neuronal WT mitochondria was smaller than that recorded for CypD-KO mitochondria, and the exact opposite was observed in astrocytes.
There are ~1500 proteins in mitochondria and less than 3% of those are mitochondrially encoded [193], while the rest are nuclear-encoded. It is at least prudent to consider that the composition and/or regulation of the mitochondrial permeability transition pore exhibits tissue or even cell-specific differences, as it has been suggested elsewhere [194;195].
DOI:10.14753/SE.2012.1675
4.2. Ca
2 +release triggered by NAADP in hepatocyte microsomes
4.2.1. NAADP induces Ca2 + release from hepatocyte microsomes
Hepatic microsomal vesicles rapidly sequestered 4 5Ca2 + in the presence of ATP (Figure 11 A), with an uptake of 4.0 ± 0.2 nmol/mg protein (n=13). The maximum of Ca2 + uptake was found within 5-10 minutes, which is slower than that observed in experiments with intact or permeabilized cells but consistent with earlier reports [196]. About 90%
of the specifically retained microsomal Ca2 + was rapidly released by ionomycin (5 µ M) (Figure 11 A). This rate of decline of microsomal Ca2 + content defined the magnitude of the microsomal Ca2 + stores available for release. We found it important to identify of the main Ca2 + transporter through which the microsomes are loaded. We determined the Ca2 + uptake of liver microsomes in the presence of 1 µ M thapsigargin, a selective inhibitor of the sarco(endo)plasmic reticulum Ca2 +-ATPase (SERCA) and 1 µ M bafilomycin A1, an established blocker of the V-type ATP-ase [197]. The Ca2 + accumulation of microsomes was nearly abolished by thapsigargin, while bafilomycin does not affect substantially the Ca2 + uptake mechanisms of liver microsomes. In the light of these results, it is the SERCA that represents the main mechanism responsible for the active loading of liver microsomes. In the next step, we investigated whether NAADP could induce Ca2 + release from rat liver microsomes loaded actively with 4 5Ca2 + and compared it to InsP3 and cADPR-induced Ca2 + release. In this assay, NAADP (10 µ M), InsP3 (10 µ M) and cADPR (10 µ M) induced a fast Ca2 + efflux, which differed significantly from control microsomes (CICR) (Figure 11 B).
The pattern of NAADP mediated Ca2 + release appeared to be biphasic, with an initial rapid release followed by a sustained but slower phase of
Ca2+ release triggered by NAADP in hepatocyte microsomes
release. A similar pattern of Ca2 + release was observed when cADPR and InsP3 were added (Figure 11 B). After 5 seconds of Ca2 + release the total
F i g u r e 1 1 . N A A D P - i n d u c e d 4 5C a2 + r e l e a s e f r o m a c t i v e l o a d e d h e p a t o c y t e m i c r o s o m e s . ( A ) T h e t i m e c o u r s e o f t h e C a2 + u p t a k e b y l i v e r m i c r o s o m e s w a s d e t e r m i n e d u s i n g 4 5C a2 +, a s d e s c r i b e d i n t h e ‘ M a t e r i a l s a n d M e t h o d s ’ s e c t i o n ( S e c t i o n 3 . 7 ) . A c c u m u l a t i o n (■) o f C a2 + w a s s t a r t e d b y a d d i t i o n o f 1 m M A T P . T h e a m o u n t o f t h e m o b i l i z a b l e C a2 + w a s d e t e r m i n e d b y a d d i n g 5 µM i o n o m y c i n ( ) t o t h e m e d i u m . T h e e f f e c t o n 4 5C a2 + u p t a k e o f 1 µ M t h a p s i g a r g i n (Δ) a n d 1 µ M b a f i l o m y c i n (□) w a s a l s o t e s t e d . B a f i l o m y c i n w a s a d d e d t o t h e m i c r o s o m e s 5 m i n u t e s b e f o r e 4 5C a2 + u p t a k e w a s i n i t i a t e d .
( B ) C o m p a r i s o n o f t h e C a2 + m o b i l i z i n g c h a r a c t e r i s t i c s o f I n s P3 (○) , c A D P R (●) a n d N A A D P (▲) ( 1 0 µ M e a c h ) . C a2 +- i n d u c e d C a2 + r e l e a s e ( C I C R ) (Δ) w a s d e t e r m i n e d b y a d j u s t i n g e x t r a v e s i c u l a r f r e e C a2 + l e v e l t o p C a = 6 u s i n g E G T A ( 1 0 0 µ M ) . R e s u l t s a r e t h e a v e r a g e ± s . e . m . o f 6 - 1 2 d e t e r m i n a t i o n o n a t l e a s t f o u r d i f f e r e n t e x p e r i m e n t a l d a y s . T h e i n s e t s h o w s t h e t o t a l a m o u n t o f C a2 + e f f l u x t r i g g e r e d b y I n s P3, c A D P R a n d N A A D P a f t e r 5 s e c o n d s o f C a2 + r e l e a s e .
( C ) M i c r o s o m e s s e q u e s t e r e d C a2 + i n t h e p r e s e n c e o f a n A T P r e g e n e r a t i n g s y s t e m ( 2 U / m l c r e a t i n e - k i n a s e , 4 m M p h o s p h o c r e a t i n e ) a n d r e l e a s e d c a l c i u m i n r e s p o n s e t o s u b s e q u e n t a d d i t i o n o f 1 0 µ M c A D P R (●) , 1 0 µ M I n s P3 (○) a n d 1 0 µ M N A A D P (▲) .
DOI:10.14753/SE.2012.1675
amount of Ca2 + efflux elicited by CICR was 0.165 ± 0.06 nmol/mg protein (4.6% of ionomycin release, n=6-12). In the same set of experiments, NAADP released 0.42 ± 0.08 nmol Ca2 +/mg protein (11.8%
of ionomycin release, n=15), while cADPR elicited 0.821 ± 0.1 nmol Ca2 +/mg protein (22.8% of ionomycin release, n=10) (Figure 11 B, inset). Under the same conditions, InsP3 released 0.7 ± 0.09 nmol Ca2 +/mg protein (19.6% of ionomycin release, n=8) (Figure 11 B, inset).
Thus NAADP is a potent, but somewhat less effective Ca2 + releasing messenger than cADPR and InsP3 in liver hepatocyte microsomes.
To further determine whether the NAADP-induced Ca2 + release mechanism in liver microsomes is distinct from the InsP3- and cADPR-mediated Ca2 + release mechanism, we tested for possible agonist cross-desensitization. As shown in Figure 11 C, we tested subsequent Ca2 + release from actively loaded liver microsomes by cADPR, InsP3 and NAADP (all applied at supramaximal concentrations, 10 µ M) in the presence of an ATP-regenerating system. NAADP managed to elicit maximal Ca2 + efflux when applied after cADPR and InsP3 have already been probed. Thus cross-desensitization to InsP3 and cADPR by NAADP did not occur (Figure 11 C). This result further supports the view that NAADP acts upon a Ca2 + release mechanism distinct from that of InsP3
and cADPR from rat liver microsomes.
4.2.2. Dose-dependence of the NAADP-mediated Ca2 + release
NAADP induced Ca2 + release in rat liver microsomes in a dose-dependent manner, with a half-maximal concentration (EC5 0) of 0.93 ± 0.1 µ M (Figure 12). Our results correspond with those of other authors who experimented with microsomes prepared from other mammalian tissues [117;118;121], whereas the EC5 0 for NAADP was reported to be one order of magnitude smaller in intact cells (in the range of 100 nM) [113;120].
Ca2+ release triggered by NAADP in hepatocyte microsomes
F i g u r e 1 2 . D o s e d e p e n d e n c e o f t h e N A A D P - i n d u c e d C a2 + r e l e a s e i n r a t l i v e r m i c r o s o m e s .
E x p e r i m e n t a l s e t t i n g s w e r e s i m i l a r t o t h o s e s h o w n i n F i g u r e 1 1 B a n d t h e d a t a a r e r e p r e s e n t a t i v e f o r f i v e i n d e p e n d e n t e x p e r i m e n t s .
DOI:10.14753/SE.2012.1675
4.2.3. Unique homologous desensitization pattern of the NAADP receptors
We investigated the inactivation phenomenon of NAADP-induced Ca2 + release in liver microsomes. First injection of subthreshold concentration of NAADP (0.1 µ M) to microsomes at the third minute during active loading did not result in substantial Ca2 + release by itself (Figure 13 A). However after 2 minutes of incubation, 10 µ M NAADP released 0.14 ± 0.04 nmol Ca2 +/mg protein compared to 0.39 ± 0.04 nmol/mg protein Ca2 + released from non pre-incubated microsomes.
On Figure 13 B we compared the dose-response curve of the NAADP-induced Ca2 + release with the curve for residual Ca2 + release by supramaximal NAADP (10 µ M) after 2 minutes pre-incubation of microsomes with different concentrations of NAADP (between 0.1 nM
F i g u r e 1 3 . U n i q u e h o m o l o g o u s d e s e n s i t i z a t i o n p a t t e r n o f t h e N A A D P r e c e p t o r s . ( A ) H o m o l o g o u s d e s e n s i t i z a t i o n o f N A A D P r e c e p t o r s b y s u b t h r e s h o l d c o n c e n t r a t i o n s o f N A A D P . A c t i v e l y l o a d e d m i c r o s o m e s (●) w e r e p r e - t r e a t e d w i t h 0 . 1 µ M N A A D P f o r t w o m i n u t e s a n d t h e n c h a l l e n g e d t o a s u p r a m a x i m a l c o n c e n t r a t i o n o f 1 0 µ M N A A D P (○) . N A A D P i n d u c e d C a2 + r e l e a s e f r o m n o n p r e - t r e a t e d m i c r o s o m e s (▲) . T h e i n s e t s h o w s t h e C a2 + e f f l u x a t f i v e m i n u t e s o f C a2 + l o a d i n g f r o m m i c r o s o m e s i n c u b a t e d b y n o n - a c t i v a t i n g c o n c e n t r a t i o n o f N A A D P a n d n o n p r e - t r e a t e d m i c r o s o m e s .
( B ) D o s e - r e s p o n s e c u r v e o f N A A D P (●) a n d t h e r e s i d u a l C a2 + r e l e a s e b y s u p r a m a x i m a l c o n c e n t r a t i o n o f N A A D P ( 1 0 µ M ) a f t e r 2 m i n p r e i n c u b a t i o n w i t h c o n c e n t r a t i o n s o f N A A D P b e t w e e n 0 . 1 n M a n d 1 0 µ M (○) .
Ca2+ release triggered by NAADP in hepatocyte microsomes
and 10 µ M). In this manner, NAADP may function as its own specific antagonist with an IC5 0 of 30 nM. The two curves form a U-shape as NAADP desensitize its receptors with an IC5 0 that is one order of magnitude lower than the EC5 0. Thus, we found evidence that, similarly to invertebrates [130], full desensitization of the NAADP receptors by subthreshold NAADP concentrations is possible without any need for previous substantial Ca2 + release. This phenomenon is in contrast to the self-desensitization mechanism for InsP3 and cADPR (X-desensitization) [130].
4.2.4. The effect of thapsigargin and bafilomycin A1 on the NAADP-evoked Ca2 + release in rat liver microsomes
The NAADP sensitive Ca2 + stores are insensitive to thapsigargin in sea urchin eggs [198] as well as in several intact mammalian cell types (e.g. arterial smooth muscle [147] and pancreatic acinar cells [141]), and can be localised in the lysosomal compartment [139;147] (acidic thapsigargin-insensitive pool). Therefore, it seemed important to test whether the NAADP-mediated Ca2 + release from rat liver microsomes is dependent on acidic pools. One way of interfering with organellar acidfication is to pretreat with bafilomycin A1, which is a blocker of the
The NAADP sensitive Ca2 + stores are insensitive to thapsigargin in sea urchin eggs [198] as well as in several intact mammalian cell types (e.g. arterial smooth muscle [147] and pancreatic acinar cells [141]), and can be localised in the lysosomal compartment [139;147] (acidic thapsigargin-insensitive pool). Therefore, it seemed important to test whether the NAADP-mediated Ca2 + release from rat liver microsomes is dependent on acidic pools. One way of interfering with organellar acidfication is to pretreat with bafilomycin A1, which is a blocker of the