(Received for publication, May 31, 1995; and in revised form, November 17, 1995)
From the
Mitochondria undergo at least two types of structural alteration in response to various physiological and pathophysiological stimuli. One type is nonreversible and is associated with mitochondrial lysis. The second is reversible and appears to be associated with calcium-mediated activation of a specific inner mitochondrial membrane channel. The mechanisms underlying the induction of this second alteration, termed a mitochondrial permeability transition (PT), have been the subject of a great deal of recent research. Using rat liver mitochondria, our data demonstrate that calcium-mediated PT induction can be affected by the lipid peroxidation byproducts 4-hydroxynonenal and 4-hydroxyhexenal (HHE). 4-Hydroxynonenal appears inactive at concentrations <1 uM but displays both stimulatory and inhibitory effects as part of a biphasic dose response between approximately 1 and 200 uM. In contrast, HHE consistently enhances calcium-mediated induction of the PT, even at femtomolar concentrations. The exquisite specificity and sensitivity of HHE led to further studies to examine the nature of this induction. Studies showing that HHE-mediated induction could be prevented by cyclosporin A confirmed PT involvement. Further studies showed that induction was dependent on both calcium and electron transport chain function. Pretreatment of the HHE with glutathione also prevented PT induction, but simultaneous addition of the thiol reagents dithiothreitol or glutathione, which often prevents PT induction, was ineffective, attesting to the effectiveness of HHE as an inducer. Together, these data provide a possible mechanistic explanation for the previously observed effects of lipid peroxidation on PT induction.
Exposure to oxidants or to phosphate, especially in the presence
of calcium, has long been known to lead to mitochondrial swelling and
damage. In the past 15 years it has become increasingly appreciated
that this swelling is due to the induction of a specific proteinaceous
channel now termed the permeability transition pore
(PTP)()(1, 2) . Induction is reversible,
but while open the channel allows free passage of all solutes under
1500 daltons, thus destroying the proton gradient and preventing
oxidative phosphorylation. Channel induction can also lead to a
potentially lethal efflux of mitochondrially sequestered calcium into
the cytoplasm of the cell.
In vitro, induction of the
channel may be observed in the presence of calcium. The addition of
phosphate (3) or oxidants such as tert-butyl
hydroperoxide (t-BuOOH) (4) greatly enhances the rate
of induction. Induction may also be achieved by respiratory
uncouplers(5) , some fatty acids (e.g. palmitoyl
CoA(2, 6) , and thiol cross-linkers such as
phenylarsine oxide(7) . Induction may be inhibited by
cyclosporin A(8, 9) , and this inhibition serves as a
specific marker for PTP induction. Channel opening by some inducers may
also be inhibited by other compounds such as magnesium (believed to
compete with calcium)(10) , EGTA (by chelating calcium) (11) , respiratory inhibitors (PT induction appears to require
respiratory function)(12, 13) ,
antioxidants(14, 15) , as well as thiol reagents such
as dithiothreitol or the presence of reduced glutathione
(GSH)(16, 17) . The channel also appears regulated by
membrane potential and matrix pH(7, 18, 19) ,
and effectors of lipid structure also appear to play roles in PT
induction (e.g. phospholipase A) (20) and
its prevention (e.g. trifluroperazine)(21) .
The potential lethality of PT induction, coupled with the apparent roles of phosphate, calcium, and oxidants in this induction, have suggested to many that activation of this pore may play roles in ischemia reperfusion injury(1, 22) . Initial support for this concept comes from long standing observations of swollen mitochondria in ischemia reperfusion injury and the observation that free radical scavengers and anti-oxidants exhibit partial protective activity. More recently, direct support has come from studies of the protective activity of PT inhibitors in models of anoxia, hypoxia, and ischemia reperfusion injury(1, 22) . Together, these studies suggest that the synergistic effects of oxidants and calcium on PT induction appear to represent a major way in which free radicals and uncontrolled calcium can damage cellular homeostasis.
Another major
way in which calcium and oxidative stress impact on cellular physiology
is through the induction of lipid peroxidation reactions. Lipid
peroxidation, via its chain reaction processes, serves to propagate and
amplify oxidant-mediated damage. Lipid peroxidation reactions have been
associated with acute pathologic occurrences (e.g. ischemia
reperfusion injury) (23) and chronic pathological conditions (e.g. diabetes) (24) , ()as well as the
aging process(26) . It is currently believed that the
byproducts of lipid peroxidation reactions, including malondialdehyde
and the hydroxyalkenals, mediate many of the detrimental effects
associated with lipid peroxidation reactions(27) .
Although malondialdehyde has been considered as the major deleterious lipid peroxidation byproduct, much of the current work on the toxicity of lipid peroxidation byproducts focuses on the more reactive hydroxyalkenals, such as 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE)(27) . These products are readily generated in vivo following application of various biological/biochemical insults. These compounds are highly reactive to amino acids, especially those containing thiol groups, and highly toxic to many cellular systems, such as mitochondria(27) . For example, HNE has been shown to impair both mitochondrial transcription (28) and respiration(29) , decrease mitochondrial membrane fluidity(30) , and impair function of the adenine nucleotide translocase(31) .
Consideration of the biochemical natures of HNE in conjunction with the current body of understanding of PT inducers (e.g. thiol cross-linkers, calcium, a possible role for lipid peroxidation in irreversible induction), led to the hypothesis that the hydroxyalkenals would be potent inducers of the PT. This possibility was further supported by the observed ability of HNE to inhibit the adenine nucleotide translocator, a protein whose functional status is believed to interact with the PT. In the current work, we therefore directly tested the prediction that hydroxyalkenals would induce the PT.
The ability of lipid peroxidation byproducts such as HHE and
HNE to induce a mitochondrial permeability transition was directly
addressed through comparison with well characterized systems. As most
systems that induce the PT require calcium, we tested HHE and HNE in
the presence of 25 uM Ca. As can be seen in Fig. 1, 180 uM HNE appears to reduce the induction time
of the PT compared with induction by calcium alone, but a decreased
rate of propagation leads to an overall slowing of the time required
for half-maximal induction. In contrast, 60 uM HHE speeds up
calcium-mediated induction by about 30%. We addressed of each of these
products separately.
Figure 1: HHE enhances calcium-mediated PT induction. Incubations were as described under ``Materials and Methods.'' The additions for specific traces were as noted. Trace a, 25 uM calcium (as calcium chloride); trace b, 25 uM calcium, 180 uM HNE; trace c, 25 uM calcium, 60 uM HHE.
HNE dose response analysis (Fig. 2) revealed that HHE displays a complex biphasic dose response curve. It is inactive at concentrations <1 uM. In the preparation shown in Fig. 2, we noted that 1.8 uM and 180 uM HNE enhanced calcium-mediated PT induction, whereas 18 uM and 60 uM inhibited it. The general form of this dose response appears consistent, but the absolute values at which the noted effects occur is variable (e.g. compare Fig. 1, trace a, with Fig. 2A, trace A). The remainder of Fig. 2shows that the effect of HNE is mediated through effects on the rate of propagation. Fig. 2B shows how the slope (rate of propagation) relative to that obtained with calcium alone is modulated by dose. Fig. 2C shows how the time of half-maximal induction relative to that obtained with calcium alone is modulated by dose. Fig. 2D shows the tight inverse relationship between these two parameters. The regression analysis in Fig. 2E shows that the inverse of the slope is indeed a strong predictor of the overall time to half-maximal induction. In the dose response curve shown in Fig. 2, lag also appears correlated, but note that Fig. 1(trace B) shows that this is not always the case. Thus, although alterations in lag may be associated with overall response to HNE, they appear to play minor roles in its effects on PT induction kinetics.
Figure 2: HNE effects on PT induction. A, trace A, 25 uM calcium, 180 uM HNE; trace B, 25 uM calcium, 60 uM HNE; trace C, 25 uM calcium, 18 uM HHE; trace D, 25 uM calcium, 6 uM HNE; trace E, 25 uM calcium, 1.8 uM HNE; trace F, 25 uM calcium, 600 nM HNE, identical with 25 uM calcium alone. B, dose response analysis of the slope of trace through the central portion of the propagation phase. The slope of trace from 25 uM calcium is defined as slope = 1. C, dose response analysis of the time required for half-maximal induction (likely representing induction of PT in 50% of the mitochondria in the sample) expressed relative to induction time in calcium alone. D and E, comparison and analysis of the data in B and C.
HHE dose response analysis (Fig. 3)
revealed that HHE enhanced calcium-mediated PT induction at
concentrations as low as 60 fM. Enhancement was regularly seen
at femtomolar concentrations (n = 9 of 9). Attomolar
concentrations were also occasionally seen to increase PT induction
(not shown), but this result was not consistent. Analysis of
HHE-mediated PT induction showed two distinct phases. At concentrations
in the micromolar range, HHE consistently reduces lag time (Fig. 1, trace c, and Fig. 3, trace B)
but has a variable effect on slope (compare Fig. 1, trace
c, with Fig. 3, trace B), which leads to a marked
variability in the time until maximal induction. This variability at
high doses makes determination of a true EC for HHE both
impractical and uninformative. In contrast to both this data and the
data on HNE presented above, submicromolar concentrations of HHE act
predominantly or exclusively by decreasing lag time (Fig. 3, B and C).
Figure 3: HHE effects on PT induction. A, trace A, 25 uM calcium; trace B, 25 uM calcium, 60 uM HHE; trace C, 25 uM calcium, 60 nM HHE; trace D, 25 uM calcium, 60 pM HHE; trace E, 25 uM calcium, 60 fM HHE (overlaps trace D). B, dose response analysis of the slope of trace through the central portion of the propagation phase. C, dose response analysis of the time required for half-maximal induction (likely representing induction of PT in 50% of the mitochondria in the sample).
HHE-mediated induction was next compared with other effectors of PTP opening. As shown in Fig. 4A, HHE-mediated effects on PT induction by nanomolar and femtomolar HHE, although consistently more rapid than calcium alone, are slower than induction by either 2 mM inorganic phosphate or 75 uMt-BuOOH. As pointed out above, however, induction by higher HHE concentrations (e.g. 60 uM) is extremely sample-specific and may be nearly as rapid as phosphate normally is (e.g.Fig. 3, trace B) or roughly the level of enhancement seen with t-BuOOH (e.g.Fig. 1and 4B). Note the different effects on lag and slope in Fig. 1and Fig. 4B, suggesting that the effects of micromolar concentrations of HHE may also differ from sample to sample.
Figure 4:
HHE
compared with known inducers of the PTP. A, trace a,
25 uM calcium; trace b, 25 uM calcium, 2
mM NaHPO
; trace c, 25 uM calcium, 75 uMt-BuOOH; trace d, 25
uM calcium, 60 nM HHE; trace e, 25 uM calcium, 60 fM HHE. B, trace a, 25 uM calcium; trace b, 25 uM calcium, 750 uMt-BuOOH; trace c, 25 uM calcium, 60 fM
HHE.
We next began characterization of
the HHE-mediated response through the use of compounds known to slow or
prevent PT induction. To confirm that response to HHE represented PT
induction, we examined the ability to block induction with cyclosporin
A, a well characterized specific inhibitor of the PT. As shown in Fig. 5, cyclosporin prevents the loss of absorbance induced by
HHE. To further evaluate factors involved in HHE-mediated PT induction,
we examined the ability of several compounds to affect HHE-mediated
induction. EGTA, myxothiazol, and antimycin A (data not shown,
identical to myxothiazol) were all able to inhibit PT induction by
Ca and HHE (Fig. 5), but glutathione (Fig. 6) and dithiothreitol (2 mM, data not shown,
identical to glutathione) were not. In contrast, pretreatment of HHE
with glutathione did prevent induction (Fig. 6). Because of the
variability observed with micromolar concentrations of HHE, EGTA,
cyclosporin, and myxothiazol were tested against 60 nM HHE.
Because these inhibitors were effective against this concentration,
lower HHE doses were not tested. In the glutathione experiment, the use
of femtomolar HHE allowed us to determine if GSH could block induction
by even the lowest effective HHE dose, which, as seen in Fig. 6,
it could not.
Figure 5: HHE-mediated PT induction is inhibited by cyclosporin, EGTA, and myxothiazol. Trace A, 25 uM calcium; trace B, 25 uM calcium, 60 nM HHE; trace C, 25 uM calcium, 3 ng/ml myxothiazol; trace D, 25 uM calcium, 60 nM HHE, 3 ng/ml myxothiazol; trace E, 25 uM calcium, 500 nM cyclosporin A; trace F, 25 uM calcium, 60 nM HHE, 500 nM cyclosporin A; trace G, 25 uM calcium, 500 uM EGTA; trace H, 25 uM calcium, 60 nM HHE, 500 uM EGTA.
Figure 6: Preincubation with glutathione inactivates HHE. Trace a, 25 uM calcium, 60 fM HHE. Trace b, 25 uM calcium, 60 fM HHE, 100 uM glutathione. In trace b, the glutathione was added to the sample prior to the addition of HHE. Trace c, 25 uM calcium, 60 fM HHE, 100 uM glutathione. In trace c, the glutathione and HHE were preincubated for 15 min at room temperature (6 µl of 10 pM HHE, 10 µl of 10 mM GSH, 10 µl of the buffer used in the PT assay) prior to addition to the reaction. Trace d, 25 uM calcium.
The polyunsaturated fatty acids that comprise biological macromolecules are extremely sensitive to oxidant-mediated damage. Although much early work focused on the damage done to the lipids themselves, more recent work has also addressed the roles that the byproducts of these peroxidation reactions play in ongoing cellular function and dysfunction.
Because of its prevalence and its ability to cross-link biomolecules, malondialdehyde was the focus of initial studies on the effects of lipid peroxide byproducts. As understanding of the field grew, however, more emphasis began to be placed on the hydroxyalkenals, especially HNE (27) . HNE is produced in during lipid peroxidation reactions from the breakdown of linoleic acid, arachidonic acid, and 15-hydroperoxyarachidonic acid(27, 33, 34) ; it is far more reactive than malondialdehyde, often impairing biological function in the low micromolar range. HHE is similarly reactive and can be formed through both lipid peroxidation (by the degradation of omega 3 polyunsaturated fatty acids, e.g. 22:6) and through nonperoxidative mechanisms, such as the metabolism of the alkaloid senecionine(27, 35, 36, 37) . Interestingly, the mechanism underlying senecionine poisoning has been shown to be HHE-mediated disruption in calcium homeostasis (38) .
It is also interesting to note the comparison between our observations and the commonly held belief that the chemical similarity between HHE and HNE means that they are also biologically similar. HHE, which is only recently beginning to be studied in detail, enhanced calcium-mediated induction of the PT at femtomolar concentrations. Higher levels of HHE (micromolar) can give extremely rapid induction, but for reasons we have been unable to determine, effects are highly variable. These observations are especially interesting given the known effect of HHE to disrupt calcium homeostasis(38) .
In contrast to HHE, the effects of HNE are
extremely dose-dependent, thus indicating a reactivity distinct from
that evidenced by HHE. For example, HNE can slow induction at
10-100 uM, whereas no inhibitory effects have ever
been observed with HHE at micromolar concentrations (data not shown).
Notably, Richter and Meier (25) have shown that concentrations
of HNE that we find slow PT induction (
10-100 uM)
can prevent pro-oxidant-induced calcium release from mitochondria,
apparently by interfering with pyridine nucleotide
hydrolysis(25, 27) , which can favor PT
induction(1) .
Inhibitors of the PT were used to further probe the mitochondrial alterations induced by HHE and calcium. The cyclosporin A studies confirmed that we were indeed studying induction of PT, as opposed to nonspecific swelling. The EGTA studies demonstrate that calcium is required for PT induction by HHE, as it is for most other inducers. The studies with respiratory inhibitors (e.g. myxothiazol, antimycin A) confirmed the role of respiration in PT induction (probably through a free radical-mediated mechanism).
The failure of both GSH and dithiothreitol to protect when added just prior to addition of HHE was unexpected, as the major known chemical activity of HHE is as a thiol-oxidant, and both GSH and dithiothreitol have been previously demonstrated to prevent PT induction by blocking oxidation of a critical thiol. Coupled with the consideration that neither endogenous mitochondrial GSH (millimolar concentrations versus femtomolar HHE concentrations) nor exogenous GSH added prior to HHE prevented PT induction by HHE, these data show that HHE is far more reactive with the channel than with GSH and that GSH is thus relatively inactive in protecting mitochondria from HHE-mediated damage. Evidence has suggested that the hydroxyalkenals such as HHE and HNE have approximately equal reactivity to GSH and have approximately a 2-min half-life in the mitochondria. This relatively long half-life in the presence of vast excess of GSH is due to the relatively low frequency of the form of GSH (GS-) (27) that reacts with the hydroxyalkenals. Thus, if the channel is commonly in a state in which it can react with HHE, the critical reactions may be far less unlikely than they seem at first. Studies showing that 15-min pretreatments of HHE with GSH prior to the addition of this reaction mixture to the mitochondria provided evidence supporting this kinetic explanation.
Our data further understanding of the observations of Richter and Meier (25) on HNE-mediated effects on mitochondria and help to explain the earlier observations on the toxicity of HHE and senecionine(38) . Beyond this, the observations that HHE and HNE can alter the effects of calcium on PTP function raises the question of the implication of this interaction between calcium and hydroxyalkenals in vivo. Certainly HHE can reach femtomolar levels in vivo, and higher concentrations of both HHE and HNE may exist locally, especially during times of increased oxidative stress. Because ischemia reperfusion injury is associated with both calcium influx and free radical-generated lipid peroxidation, our data provide a potentially testable mechanism for the putative role of PT induction in ischemia reperfusion injury(1, 22) . This interaction may become even more important should cellular and mitochondrial defenses become compromised due to progressive age or disease status.
In conclusion, it is important to note that the potent effects of HHE on the PT suggest that the time has come to focus more attention on the lipids that comprise the inner mitochondrial membrane and how these lipids may play roles in PT induction. Free radical-mediated lipid peroxidation and the associated production of both HHE and HNE may affect PT function during aging, chronic disease, and acute events such as ischemia reperfusion injury. Membranes may also affect PT function in other ways, because alterations in membrane structure and function, such as those that occur with age, may alter calcium balance or membrane potential, both of which can affect the PT.