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Vol. 54, Issue 1, 101-127, March 2002
Laboratory of Intracellular Ion Channels (A.S.) and Laboratory of Bioenergetics, Biomembranes and Metabolic Regulations (L.W.), Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland
Abstract
I. Introduction
II. Mitochondria and the Cell
III. Mitochondria in Chemotherapy-Induced Apoptosis
A. The Mitochondrial Pathway of Apoptosis
B. Mitochondria as Targets in the Control of Apoptosis
C. Antitumor Drugs as Apoptosis Promoters
IV. Mitochondria and Oxidative Stress, Aging, and Degenerative Diseases
V. Interaction of Potassium Channel Openers with Mitochondria
A. Potassium Channel Openers and Mitochondrial K+ Channels
B. Mitochondrial ATP-Regulated Potassium Channel: A Novel Effector of Cardioprotection
VI. Sulfonylureas and Mitochondria
A. Functional Effects of Antidiabetic Sulfonylureas on Mitochondria
B. Effect of Antitumor Sulfonylureas on Mitochondria
VII. The Mitochondrial Benzodiazepine Receptor
VIII. Immunosuppressant Drugs and Mitochondria
IX. Disruption of Mitochondrial Functions by Antiviral Drugs
X. Nonsteroidal Anti-Inflammatory Drugs and Mitochondria
XI. Local Anesthetics and Mitochondrial Energy Metabolism
XII. Mitochondria as a Pharmacological Target of Lipid Metabolism
A. Inhibition of the Transfer of "Activated" Fatty Acids into Mitochondria and of Their-Oxidation
B. L-Carnitine Supplementation
C. Nonesterified Fatty Acids as "Natural" Uncouplers: Role in Thermogenesis and Obesity Control
D. N-Acylethanolamines
XIII. Final Remarks
Acknowledgments
References
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Abstract |
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Mitochondria play a central role in energy metabolism within the cell. Mitochondrial dysfunctions lead to various neurodegenerative disorders and to the so-called "mitochondrial diseases". A vast amount of evidence points to the implication of mitochondria in such complex processes as apoptosis and cardioprotection. The purpose of this review is to present a recent state of our knowledge and understanding of the action of various therapeutically applied substances on mitochondria. These include antitumor, immunosuppressant, and antiviral drugs, potassium channel openers, sulfonylureas, and anesthetics. Some of these substances are specifically designed to affect mitochondrial functions. In other cases, drugs with primary targets in other cellular locations may modify mitochondrial functions as side effects. In any case, identification of mitochondria as primary or secondary targets of a drug may help us to better understand the drug's mechanism of action and open new perspectives for its application. As far as possible, the molecular mechanisms of the interference of particular drugs in the mitochondrial metabolism will be described. In some cases, metabolic routes in which the drugs interfere will also be briefly outlined.
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I. Introduction |
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Mitochondria play a central role in energy-generating processes within the cell. Apart from this important function, mitochondria are involved in such complex processes as apoptosis and cardioprotection. A rapidly expanding body of literature also suggests that mitochondrial dysfunctions play pivotal roles in neurodegenerative disorders ranging from Parkinson's to Huntington's to Alzheimer's diseases. Mitochondrial DNA mutations, whether inherited or acquired, cause impaired respiratory chain functioning. This, in turn, leads to decreased production of ATP, formation of free radicals, and alterations in cellular calcium handling. These events may initiate peroxidation of mitochondrial DNA, proteins, and lipids, and opening of the mitochondrial permeability transition pore, an event linked to apoptotic cell death. Mitochondria are also targets for drugs such as antidiabetic sulfonylureas, immunosupressants, some antilipidemic agents, etc. The aim of this review is to present interactions of various therapeutically applied substances with mitochondria as their primary or secondary targets. To make the mechanisms of these effects comprehensible, we will also briefly describe the metabolic routes in which the drugs in question interfere.
Medically applied substances that interact with mitochondria can be divided into two groups: 1) those that are specifically designed to affect mitochondrial functions, and 2) those for which primary targets are other cellular locations and their interactions with mitochondria are secondary. In any case, identification of mitochondria as primary or secondary targets of a drug may facilitate a better understanding of its mechanism of action and open new perspectives of its application. For example, recent studies on the cardioprotective action of potassium channel openers have revealed that cardiac mitochondria are more important as the primary targets of these drugs than is the plasma membrane. Recognition of drug interaction with mitochondria as secondary targets may help us to understand the mechanisms of side effects and to construct new drugs in which these side effects will be eliminated or minimized.
Mitochondria can also be affected by a number of toxins, especially
those that interact with their respiratory and ATP-generating functions. This field is the subject of a recent review by Wallace and
Starkov (2000)
and will not be dealt with in the present article.
When dealing with particular classes of pharmaceuticals, we will often refer to original experimental work in which the drug is used in concentrations that may exceed those encountered under therapeutic conditions, especially for compounds interacting with mitochondria as their secondary targets. We consider such studies useful because they help us to understand the mechanisms of potential side effects, especially under conditions of chronic administration or overdose. In addition, some drugs may accumulate in particular tissues or organs to attain concentrations higher than those calculated for the whole body. Moreover, mitochondria are unique cellular organelles with alkaline and negatively charged interior, conditions that promote accumulation of some compounds.
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II. Mitochondria and the Cell |
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The number of mitochondria per cell is roughly related to cell energy demands. Somatic tissues contain from a few dozen to several thousand mitochondria per cell. Human spermatozoa contain a fixed number of 16 mitochondria and oocytes up to 100,000. Organs that are very active metabolically, such as muscles, liver, brain, and cardiac and skeletal muscles, contain the largest number of mitochondria and are most susceptible to drugs acting on mitochondria and to mitochondrial pathologies.
In major mammalian tissues, 80 to 90% of ATP is generated by
mitochondria in the process of oxidative phosphorylation. The mitochondrial respiratory chain, located in the inner mitochondrial membrane, is composed of enzymes and low molecular weight redox intermediates (coenzymes) that transport "reducing equivalents", in
fact hydrogen atoms or just their electrons, from respiratory substrates to molecular oxygen, down the redox potential. This hydrogen/electron current forms three cascades in which the redox energy is high enough to be utilized to extrude protons from the mitochondrial inner compartment, the matrix, to the intermembrane space. The electrochemical proton gradient thus formed, also designated as the protonmotive force
p, is the driving force for the back flow
of protons through the ATP synthase complex (Fig.
1B). This gradient is composed of the
electric component
(
2) and the
proton concentration gradient. As a result, mitochondria are unique
cellular organelles that can build up a transmembrane electric
potential of up to 180 mV, negative inside, and whose internal milieu
maintains a pH value of about 8 (Nicholls and Ferguson, 1992
). As a
consequence, they can not only accumulate membrane-permeable compounds
of cationic character, but also trap weak acids in their anionic form.
Both properties may be of importance in targeting specific drugs into
mitochondria (see, for example, Section III.C.).
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Because of a vital role of mitochondria in cell metabolism, a constant
and intense flux of inorganic ions and metabolites occurs between the
cytosol and mitochondria. Due to the presence of a pore protein, termed
porin or voltage-dependent anion channel (VDAC) (Mannella et al.,
1992
), the outer mitochondrial membrane is permeable to polar molecules
of up to 5 kDa. In contrast, the inner membrane is freely permeable to
just a few compounds such as water, O2,
CO2, and NH3. Other
hydrophilic metabolites and all inorganic ions of biological importance
can cross the membrane due to the presence of specific channels and
carrier proteins. Among the latter ones of special importance are
carriers for phosphate (Pi), adenine nucleotides
ADP and ATP, and the respiratory substrates mono-, di-, and
tricarboxylates. In general, these transport proteins operate in the
exchange mode, i.e., ADP is exchanged for ATP, Pi
for OH
, dicarboxylic anion for
Pi anion, etc. (Palmieri et al., 1992
; Kuan and
Saier, 1993
; Palmieri, 1994
; Sluse, 1996
).
Channels selective for major inorganic cations
K+, Na+,
Mg2+, and Ca2+ have been
identified in mitochondria (Bernardi, 1999
). Of particular importance
are channels for K+ that will be dealt with later
in this review (Section V.). Apart from these
channels, which must be regulated in a subtle way to prevent collapse
of the membrane potential, there are several cation exchangers, e.g.,
for K+/H+,
Ca2+/H+, and
Ca2+/Na+ exchange
(Bernardi, 1999
). An inner membrane anion channel has also been
described (Beavis, 1992
), but its molecular identity, characteristics,
and control are less well recognized. Recently, a mitochondrial
chloride channel has been cloned (Fernández-Salas et al., 1999
).
Many lipophilic compounds penetrate the inner mitochondrial membrane
freely. Among them, the most important are fatty acids. Undissociated
molecules of long-chain fatty acids can easily penetrate the membrane
in a "flip-flop" mode (McLaughlin and Dilger, 1980
; Gutknecht,
1988
). In contrast, a spontaneous crossing of the phospholipid bilayer
by fatty acid anions is extremely slow (Kamp and Hamilton, 1992
)
because of the negatively charged polar carboxylic group. However, in
the inner mitochondrial membrane, the transmembrane passage of fatty
acid anions is facilitated, probably in an unspecific way, by several
mitochondrial anion carrier proteins. This is a process that is
essential for the protonophoric action of fatty acids in mitochondria
(Wojtczak and Wi
ckowski, 1999
). Many lipophilic and
amphiphilic drugs are good mitochondrial penetrants.
Although the outer and the inner mitochondrial membranes are well
defined structures, each of them possessing different sets of enzymes
and fulfilling different functions, intimate contacts between the two
membranes have been identified on both morphological and functional
grounds (Brdiczka, 1991
).
Mitochondria contain circular DNA that encodes about two dozen
polypeptide chains participating, as subunits, in mitochondrial respiratory chain complexes and other essential components of the
energy-coupling machinery. However, the majority of mitochondrial proteins are encoded by nuclear DNA and synthesized outside the mitochondria. They are imported into these organelles by a complex multistep mechanism (Lill and Neupert, 1996
; Schatz, 1996
).
Although the end product of the respiratory chain is water that is
generated in a four-electron reduction of molecular oxygen by
cytochrome oxidase (complex IV), a minor proportion of
O2 can be involved in one-electron reduction
processes, generating so-called "reactive oxygen species" (ROS), in
particular, superoxide anion radical
O
). Some contribution of complex I to this
process has also been found. Reactive oxygen species are generally
regarded as toxic metabolites and, as such, are decomposed by
specialized enzymes: catalase, peroxidases, and superoxide dismutases.
Nevertheless, a part of these reactive compounds that has sustained
this catalytic removal may have a dramatic effect on mitochondria and
the cell as a whole by eliciting a cascade of events leading to
programmed cell death, so-called "apoptosis" (see
Section III.).
This brief description of mitochondrial functions is aimed to point to multiple pathways interconnecting mitochondrial metabolic routes with those of the rest of the cell. Pharmacological agents, which affect either intramitochondrial metabolic processes or transport pathways connecting mitochondria with the cytosol, may therefore have a considerable influence on the total cell metabolism. Figure 1A presents the main mitochondrial processes and their locations within various mitochondrial compartments as well as routes of communication between them and the rest of the cell. It will also help to locate processes and points of pharmacological attack that will be dealt with in subsequent sections.
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III. Mitochondria in Chemotherapy-Induced Apoptosis |
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Programmed cell death, or apoptosis, is a common
process in multicellular organisms. It enables the elimination of
single cells or their assemblies when their natural biological function has come to an end or when the cell has become damaged or mutated to
such an extent that its further existence might be dangerous to the
whole organism. In particular, apoptosis occurs in embryogenesis, in
metamorphosis, and in the growth and maturation of individual organs.
Being of such vital importance, apoptosis has attained, during
evolution, a high degree of genetic and metabolic control. This broad
subject is covered by numerous excellent reviews (e.g., Ellis et al.,
1991
; Kroemer et al., 1995
; Vaux and Strasser, 1996
; Hengartner, 2000
).
Therefore, the present considerations will be limited to the function
that is played in apoptosis by mitochondria and to the pharmacological
modulations of apoptosis in relation to cancer therapy (for a
comprehensive review, see also Deigner and Kinscherf, 1999
). It is
generally believed that transformations that may lead to malignancy are
rather common in cells of highly organized animals, including humans.
However, such cells, as a rule, are efficiently eliminated by apoptosis
put in operation by mechanisms deeply encoded in their genome. Only
cells that have escaped those rescue systems give rise to malignant growth.
A. The Mitochondrial Pathway of Apoptosis
In general, two partly interdependent routes may lead to apoptosis
(Hengartner, 2000
; Kaufmann and Earnshaw, 2000
). One of them is
initiated by ligation of the so-called death receptors at the cell
surface (Ashkenazi and Dixit, 1999
), whereas the other one involves
mitochondria (Petit et al., 1997
; Green and Reed, 1998
; Kroemer et al.,
1998
; Mignotte and Vayssiere, 1998
; Susin et al., 1998
; Kroemer, 1999
;
Desagher and Martinou, 2000
; Halestrap et al., 2000
). In the latter
case, one of the early events leading to apoptosis is the release of
cytochrome c from mitochondria (Liu et al., 1996
; Kluck et
al., 1997
; Reed, 1997
; Yang et al., 1997
; Martinou et al., 2000
). Along
with another mitochondrial protein, the apoptosis-inducing
factor (Daugas et al., 2000
), it elicits in the cytosol a
cascade of events leading to the activation of intracellular proteases
of the caspase family (Earnshaw et al., 1999
) and, eventually, to a
partial self-digestion of the cell (Bossy-Wetzel and Green, 1999
) (Fig.
2). The mechanism by which cytochrome
c is liberated from mitochondria to the cytosol is
debatable. According to some authors (e.g., Scarlett and Murphy, 1997
;
Vander Heiden et al., 1997
; Petit et al., 1998
), this is preceded by
mitochondrial swelling that leads to disruption of the outer membrane.
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More recent reports indicate, however, that cytochrome c is
liberated from mitochondria by special mechanisms under conditions of
preserved intactness of the outer membrane (Jürgensmeier et al.,
1998
; Doran and Halestrap, 2000
). A decisive role in this process is
played by the mitochondrial permeability transition pore (PTP) and the
proapoptotic protein Bax (Marzo et al., 1998
; Crompton, 1999
). PTP is
located in the contact sites between the outer and the inner
mitochondrial membranes and, in its open state, enables a free passage
of low molecular weight compounds, up to molecular weight 1500, between
the mitochondrial inner compartment (matrix) and the cytosol. It
constitutes a complex assembly of porin, adenine nucleotide
translocase, and cyclophilin D (Fig. 3).
The opening of PTP is favored by factors such as
Ca2+ accumulation in mitochondria (Hunter et al.,
1976
), prooxidants (Byrne et al., 1999
), and low mitochondrial
transmembrane potential (Bernardi, 1992
) (for more information see the
reviews by Zoratti and Szabó, 1995
; Bernardi, 1996
; Bernardi et
al., 1998
; Fontaine and Bernardi, 1999
; Halestrap, 1999
).
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PTP alone is too small to enable the release of cytochrome c
(13 kDa). However, its association with Bax forms a channel specific for cytochrome c and apoptosis-inducing factor (Hirsch et
al., 1997
; Narita et al., 1998
; Jacotot et al., 1999
; Murphy et al., 2000
). Association of Bax with PTP and its pore-forming activity are
prevented by the antiapoptotic protein Bcl-2 (Kluck et al., 1997
; Yang
et al., 1997
; Murphy et al., 2000
). Thus, a subtle balance between
these two (and other similar) proapoptotic and antiapoptotic proteins
and their interaction with PTP are decisive for the survival or
apoptotic death of the cell. This balance can be affected by a number
of mitochondria-targeted drugs.
B. Mitochondria as Targets in the Control of Apoptosis
Because of the pivotal role that mitochondria play in initiating
apoptotic cell decay, they are a vulnerable target for experimental and/or pharmacological intervention. As mentioned above, one of the
main physiological factors that regulates the open probability of PTP
is calcium ion. Therefore, the role of Ca2+ in
the regulation of apoptosis has attained much attention, but the
picture still remains obscure. Numerous observations have revealed an
increased cytosolic Ca2+ concentration during
apoptosis (McConkey and Orrenius, 1997
). Manipulation of
Ca2+ concentration within the intact cell can be
performed, for example, by using thapsigargin, a potent inhibitor of
endoplasmic reticulum Ca2+-ATPase (Thastrup et
al., 1990
), which prevents calcium accumulation in the endoplasmic
reticulum. It has been observed (Zhu and Loh, 1995
; Waring and Beaver,
1996
; Bian et al., 1997
) that thapsigargin induces apoptosis in various
cell lines. Apoptosis induced in thymocytes by very low concentrations
of thapsigargin can be prevented by cyclosporin A (Waring and Beaver,
1996
), the well know inhibitor of mitochondrial PTP (Broekemeier et
al., 1989
), thus pointing to the involvement of mitochondria in this
process. On the other hand, it has been observed that thapsigargin also
initiates apoptosis after removal of extracellular
Ca2+ (Bian et al., 1997
), i.e., under conditions
in which accumulation of calcium ions inside the mitochondria is
prevented. Zhu et al. (2000)
have recently come to the conclusion that
mitochondrial Ca2+ depletion promotes apoptosis,
whereas mitochondrial Ca2+ overload leads to
necrosis of cardiac myocytes and neuroblastoma cells. Thus, the role of
mitochondria as a target for calcium ions in the regulation of
apoptosis still awaits its elucidation.
Another apoptosis-promoting factor is ROS (Buttke and Sandstrom,
1994
; Slater et al., 1995
; Rollet-Labelle et al., 1998
; Jabs, 1999
;
Chandra et al., 2000
; Matés and Sánchez-Jiménez,
2000
). Excessive production of ROS in the cell can be induced by a
number of xenobiotics, transition metal ions, and ultraviolet and
ionizing radiations (Halliwell and Gutteridge, 1989
, 1990
). ROS action on mitochondria results in the opening of PTP (Kowaltowski et al.,
1996
; Vercesi et al., 1997
) and thus triggers the mitochondria-related apoptotic pathway. It is also possible that peroxidative attack may
directly damage the outer mitochondrial membrane, resulting in
unspecific liberation of intermembrane proteins, including a fraction
of cytochrome c. Apoptosis induced by ultraviolet radiation can be reduced or completely prevented by glutathione (Slyshenkov et
al., 2001
) that removes oxygen free radicals. This observation indicates that ultraviolet radiation initiates apoptosis by acting directly on mitochondria rather than on the genomic system. Ionizing radiation (X-ray and
-radiation), often used in cancer treatment, also acts by inducing apoptosis. Being more energetic than ultraviolet radiation, it also affects DNA and thus puts into operation both the
DNA- and the mitochondria-operated apoptosis pathways (Rupnow and Knox,
1999
).
Because PTP opens upon collapse of 
, chemical and physical agents
that discharge the mitochondrial membrane potential can be regarded as
pro-apoptogenic. However, the picture is complicated by the fact that a
complete deenergization of the cell leads to necrosis rather than to
apoptosis (Leist and Nicotera, 1997
). Therefore, most mitochondrial
protonophoric uncouplers do not induce apoptosis. On the other hand,
opening of PTP by other factors results in a 
decrease due to an
increased proton influx through the pore. Thus, the PTP opening can be
either the result or the causative agent of 
collapse. Having
this in mind, one has to critically evaluate a long list of anticancer
agents causing cell death and "disruption" of 
, as presented
by Decaudin et al. (1998)
.
C. Antitumor Drugs as Apoptosis Promoters
Recent studies have shown that a large number of anticancer drugs
exert their therapeutic action by inducing the apoptosis of malignant
cells (Debatin, 2000
; Preston et al., 2001
), mainly due to activation
of the cytochrome c/caspase-9 pathway (Kaufmann and
Earnshaw, 2000
). Thus, etoposide, doxorubicin,
1-
-D-arabinofuranosylcytosine (Decaudin et
al., 1997
), lonidamine (Ravagnan et al., 1999
), betulinic acid,
arsenite, CD437, and several amphiphilic cationic
-helical peptides
(Decaudin et al., 1998
; Costantini et al., 2000
) induce apoptosis of
malignant cells by activating PTP or otherwise affecting the
mitochondrial membrane. The proapoptotic action of some of these
compounds, e.g., that of lonidamine (Ravagnan et al., 1999
), is
counteracted by cyclosporin A (CsA) and prevented by overexpression of
the antiapoptotic protein Bcl-2. This points to its mechanism of action
by the opening of the PTP.
Adriamycin (doxorubicin) is a potent antitumor drug of the
anthracycline antibiotic group the proapoptotic action of which is well
documented (Müller et al., 1998
; Kumar et al., 1999
). Its
application in chemotherapy is, however, strongly limited by its well
known high cytotoxicity (cardio-, nephro-, and neurotoxicity) (Julka et
al., 1993
; Morgan et al., 1998
; Singal et al., 2000
). These toxic
effects can be alleviated by antioxidants (Singal et al., 1997
; DeAtley
et al., 1999
), thus confirming the notion (Olson et al., 1981
;
Sokolove, 1994
) that Adriamycin and other quinoid anthracyclines are
free radical generators (Fig. 2).
A novel anti-prostate cancer hydroxamic acid derivative,
cis-1-hydroxy-4-(1-naphthyl)-6-octylpiperidine-2-one
(designated as BMD188) (Tang et al., 1998
; Li et al., 1999
), is
also an apoptosis-inducing agent. Although its mechanism of action is
unclear and, at least in some cell lines, it does not involve
cytochrome c liberation from mitochondria (Tang et al.,
1998
), more recent studies have provided evidence that its target is
the mitochondrial respiratory chain and that it induces a rapid
production of ROS (Joshi et al., 1999
). An involvement of PTP seems,
therefore, very likely (Fig. 2).
An ingenious way of destroying malignant tissues by synthetic doubly
targeted peptides has been proposed (Arap et al., 1998
; Ellerby et al.,
1999
). The authors designed helical amphipathic peptides containing
mostly cationic amino acids that preferentially bound to the inner
mitochondrial membrane (and prokaryotic cytoplasmic membranes) due to
its high transmembrane potential and high content of anionic
phospholipids. This binding resulted in the distortion of the lipid
core of the membrane, manifested by mitochondrial swelling. These
peptides manifested a much lower affinity toward the eukaryotic plasma
membrane because of its lower membrane potential and content of mostly
zwitterionic phospholipids. For that reason, they affected the
integrity of the plasma membrane at concentrations hundreds of times
higher than those needed to destroy bacterial or mitochondrial
membranes. These peptides were then coupled to short cyclic peptides
designed to be targeted toward angiogenic endothelial cells and some
malignant cell lines (Pasqualini et al., 1997
; Arap et al., 1998
). Such
hybrid peptides were internalized into the cytosol of angiogenic (but
not angiostatic) endothelial cells and cancer cells, and their helical
moieties associated with mitochondria, resulting in the swelling of
these organelles. This led to classical symptoms of apoptosis. This
treatment proved successful not only in killing cells in culture but
also in hampering the growth of human breast cancer implanted into mice
and in prolonging by several months the survival of such mice (Arap et
al., 1998
; Ellerby et al., 1999
).
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IV. Mitochondria and Oxidative Stress, Aging, and Degenerative Diseases |
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The biological importance of ROS has attracted an enormous interest during recent years due to their major role, both beneficial and noxious, in numerous vital processes. This subject is covered by thousands of original research papers and hundreds of review articles appearing each year. Only some of them can be referred to in this section. We will cite, in particular, the most recent review articles, as they will lead the reader to broader original literature and the most up-to-date information.
Mitochondria are the main source of the superoxide radical and other
reactive oxygen species that may generate from them (Chance et al.,
1979
). The main mechanisms responsible for mitochondrial ROS production
are the respiratory chain, in particular its complexes I and III
(Beyer, 1992
; Cadenas and Davies, 2000
; Raha and Robinson, 2000
), in
the inner mitochondrial membrane, and monoamine oxidase in the outer
membrane. Normally, ROS are decomposed or their peroxidation products
are neutralized by natural defense systems mainly consisting of
mitochondrial (manganese-containing) and cytosolic (containing Cu and
Zn) superoxide dismutases (Mn- and CuZn-superooxide dismutase, respectively), glutathione peroxidase, and phospholipid hydroperoxide glutathione peroxidase (Neubert et al., 1962
; Fridovich, 1974
; Chance
et al., 1979
; Ursini et al., 1986
; Augustin et al., 1997
). However,
under conditions of increased ROS generation, e.g., in ischemia-reperfusion, action of some xenobiotics, inflammation, aging,
and ultraviolet or ionizing irradiation, or conditions of impaired
antioxidant defense system, ROS may accumulate, exerting a potent
damaging effect on the cell and the whole organism (Halliwell and
Gutteridge, 1989
, 1990
; Mizuno et al., 1998
; Schapira, 1999
; Cadenas
and Davies, 2000
; Raha and Robinson, 2000
). The noxious action of ROS
mainly consists of the peroxidation of lipids, in particular
phospholipids of biological membranes, and oxidative damage to proteins
and DNA (Halliwell and Gutteridge, 1990
; Lenaz et al., 1999
; Cadenas
and Davies, 2000
). In particular, the aging of animals and humans is
connected with increased mitochondrial production of ROS (Miquel et
al., 1980
; Lenaz, 1998
; Cadenas and Davies, 2000
; Lenaz et al., 2000
;
Sastre et al., 2000
; Raha and Robinson, 2000
). Mitochondria, being the
main site of ROS generation in the cell, are also their primary target.
This, in turn, results in damage to the mitochondrial respiratory chain
and, as a consequence, a further increase in ROS generation. A
vicious cycle is thus formed (Lenaz et al., 2000
) that may
be a causative agent of a number of age-associated dysfunctions of
mitochondria and also one of the mechanisms inducing programmed cell
death (see Section III.).
To protect mitochondria and the cell against such damaging effects,
several measures can be applied. One of them is increasing the
intracellular glutathione content. This can be done by supplying precursors for glutathione synthesis, e.g.,
N-acetylcysteine, which by itself has antioxidant properties
(Benrahmoune et al., 2000
). Increasing the cell content of CoA by
supplying its precursor pantothenic acid (Slyshenkov et al., 1995
) also
increases the glutathione level (Slyshenkov et al., 1996
), although the
mechanism by which an increased content of CoA accelerates glutathione
synthesis is not fully understood. Glutathione content and its
reduction state can also be increased by incubating the cells with
curcumin, the yellow pigment of the Indian spice curry (Jaruga et al.,
1998
), or with the analgesic drug flupirtine (Perovic et al., 1996
); but the mechanisms of these processes are even more obscure. These ways
of protecting mitochondria against damaging effects of ROS, although
very effective, are not specifically mitochondria-directed because the
glutathione content increases in the whole cell and not only in mitochondria.
Other compounds acting as general intracellular antioxidants are
ascorbic acid (vitamin C),
-tocopherol (vitamin E),
-carotene (Sies et al., 1992
), and
-lipoic acid (Packer et al., 1995
). All
these compounds are naturally present in the cell, but their contents
can be increased when they are additionally administered.
Ubiquinol, the reduced form of coenzyme Q, is a naturally occurring
antioxidant which can be targeted more specifically to mitochondria and
other cell membranes (Ernster, 1994
; Forstmark-Andrée et al.,
1995
; Andrée et al., 1999
; Shi et al., 1999
). The redox couple of
ubiquinone/ubiquinol plays a key role in the mitochondrial respiratory
chain as a link between its complexes I, II, and III (Nicholls and
Ferguson, 1992
). Much attention has recently been paid to ubiquinone
and ubiquinol as potential anti-aging remedies (Lenaz, 1998
; Huertas et
al., 1999
; Lenaz et al., 2000
). Some hope has also been expressed as to
the beneficial effect of coenzyme Q in retarding and/or reducing
symptoms of Alzheimer's, Huntington's and Parkinson's diseases
(Beal, 1998
; Beal et al., 1998
; Cassarino and Bennett, 1999
),
considered to be caused by a damaging action of ROS in the central
nervous system (Mizuno et al., 1998
; Schapira, 1999
).
The situation with ubiquinol as an antioxidant is, however, complicated
by the fact that its operation in the respiratory chain may also be,
under specific conditions, a source of free radicals (Beyer, 1992
; Nohl
et al., 1999
; Cadenas and Davies, 2000
; Raha et al., 2000
). In this
respect, ubiquinol resembles some other antioxidants; for example,
ascorbic acid and
-carotene, well known to both scavenge and
generate free radicals.
An ingenious way of specifically introducing antioxidants to
mitochondria within the intact cell has been recently proposed by
Murphy and coworkers (Smith et al., 1999
; Coulter et al., 2000
; Murphy
and Smith, 2000
). These authors covalently coupled antioxidant moieties
with the lipophilic triphenylphosphonium cation. Such compounds, being
positively charged, are accumulated within mitochondria up to a
thousand-fold, driven by the transmembrane electric potential of about
200 mV (negative inside) in fully energized mitochondria. Such
accumulation can be further increased by the electric potential at the
plasma membrane of 30 to 60 mV (also negative inside), so that the
calculated inside to outside concentration ratio may amount up to
104:1. Among the antioxidant moieties coupled to
the lipophilic cation were natural products vitamin E and ubiquinol,
fullerene derivatives, and a spin trap (Coulter et al., 2000
).
Tetraphenylphosphonium coupled to vitamin E appeared to exert a potent
protective effect against the damage of isolated mitochondria by a
hydroxyl radical-generating system (Smith et al., 1999
).
Recently, it has been found (Schlüter et al., 2000
) that the
nonopioid analgesic drug, flupirtine (a fluor-containing
triaminopyridine derivative), is an effective antioxidant in
mitochondria. Being positively charged, flupirtine most likely
accumulates in mitochondria where it efficiently scavenges free
radicals. This action of flupirtine explains why this compound prevents
apoptosis of cultured cells induced by oxidative stress (Lorenz et al.,
1998
) and protects rabbit retina against ischemic injury (Osborne et
al., 1996
).
Finally, it has to be mentioned that an effective way of diminishing
mitochondrial production of ROS is a partial uncoupling, i.e.,
decreasing, of the mitochondrial transmembrane potential (
) and
of the redox state of complex I of the respiratory chain (Skulachev,
1996
). This can be obtained by increasing the permeability of the inner
mitochondrial membrane to K+ or
H+ by means of the opening of mitochondrial
potassium channels (see Section V.) or by
endogenous uncoupling proteins (see Section XII.).
| |
V. Interaction of Potassium Channel Openers with Mitochondria |
|---|
|
|
|---|
Potassium channel openers (KCOs) are agents, discovered in the
early 1980s, that act by stimulating ion flux through
K+ channels (Edwards and Weston, 1993
;
Challinor-Rogers and McPherson, 1994
; Quast et al., 1994
). Many drugs
such as cromakalim, nicorandil, and diazoxide have been identified as
KCOs (Duty and Weston, 1990
; Edwards and Weston, 1995
). KCOs act on two
types of ion channels: ATP-regulated K+ channels
(KATP channels) (Ashcroft, 2000
) and
Ca2+-activated K+ channels
(BK channels) (Lawson, 2000
). KCOs were first identified by their
antianginal or antihypertensive mode of action (Edwards and Weston,
1993
). Now, they are at various stages of development as antiasthmatic
(Morley, 1994
; Buchheit and Fozard, 1999
; Prasad et al., 2000
) and
cardioprotective agents (Grover and Garlid, 2000
). Preclinical and
clinical evidence also supports the therapeutic role of KCOs in
pulmonary (Wanstall and Jeffery, 1998
) and vascular hypertension
(Quast, 1992
; Atwal, 1994
; Quast et al., 1994
; Schachter, 1995
; Lawson,
1996
), and the treatment of overactive bladder (Andersson, 2000
).
Until recently, the effects of KCOs were believed to be attributable
entirely to the modulation of K+ channels in cell
surface membranes. It is now apparent, however, that new targets for
KCOs exist in intracellular membranes including those of sarcoplasmic
reticulum (Kourie, 1998
), zymogen granules (Thevenod et al., 1992
), and
mitochondria (Garlid, 1994
; 1996
; Szewczyk et al., 1996a
; Szewczyk,
1997
). Mitochondria seem to be particularly important targets for KCOs
because the interaction of KCOs with these organelles appears to
mediate the cardioprotective action of these compounds (Szewczyk and
Marban, 1999
; Grover and Garlid, 2000
). The protective role of
mitochondrial ion channels was recently summarized in an excellent
review article (O'Rourke, 2000
). Mitochondrial targets for
anti-ischemic drugs were recently described (Morin et al., 2001
;
Suleiman et al., 2001
).
A. Potassium Channel Openers and Mitochondrial K+ Channels
A small-conductance potassium channel, with properties similar to
those of the KATP channel from the plasma
membrane, was described in the inner membrane of rat liver and beef
heart mitochondria and designated the mitochondrial ATP-regulated
potassium channel (mitoKATP channel) (Inoue et
al., 1991
; Paucek et al., 1992
). The mitoKATP
channel was blocked not only by ATP, but also, similarly to the plasma
membrane KATP channel, by antidiabetic
sulfonylureas (Fig. 4) (Inoue et al.,
1991
; Paucek et al., 1992
). These observations raised the question
whether the mitoKATP channel could be activated by KCOs. In fact, an increased influx of K+ and
depolarization of liver mitochondria in the presence of KCOs such as
RP66471 was observed (Szewczyk et al., 1993
, 1995
). Also, other KCOs
were shown to activate potassium ion transport into mitochondria
(Belyaeva et al., 1993
; Czy
et al., 1995
; Garlid et al.,
1996b
; Holmuhamedov et al., 1998
). Moreover, ATP-inhibited K+ flux was restored by diazoxide
(K1/2 of 0.4 µM), cromakalim
(K1/2 of 1 µM), and two cromakalim
analogs, EMD60480 and EMD57970 (K1/2 of 6 nM) (Garlid et al., 1996b
). KCOs such as pinacidil, cromakalim, and levcromakalim have been shown to depolarize cardiac mitochondria (Holmuhamedov et al., 1998
). KCO-induced membrane depolarization was
associated with an increase in the rate of mitochondrial respiration and decreased ATP synthesis (Holmuhamedov et al., 1998
). Moreover, KCOs
released calcium ions and cytochrome c from cardiac
mitochondria (Holmuhamedov et al., 1998
). Despite the effect on
K+ transport, diazoxide also exhibits a direct
effect on mitochondrial energy metabolism by inhibition of respiratory
chain complex II in liver mitochondria (Grimmsmann and Rustenbeck,
1998
). Recently, mitoKATP channel opener
BMS-191095 with no peripheral vasodilator activity was described
(Grover et al., 2001
).
|
Using isolated mitochondria or proteoliposomes reconstituted with
partly purified mitoKATP channel and measuring
potassium flux, Garlid et al. (1996b)
demonstrated that heart and liver mitoKATP channels share some pharmacological
properties with the plasma membrane KATP channel,
i.e., they are both activated by KCOs. The sensitivity of cardiac
mitoKATP channels to diazoxide appeared to be
1000 times higher than that of plasma membrane KATP channels. This observation established that
the interaction of KCOs with mitoKATP channels
plays an important role in cardioprotection.
B. Mitochondrial ATP-Regulated Potassium Channel: A Novel Effector of Cardioprotection
KCOs mimic cardiac ischemic preconditioning in the absence of
ischemia, whereas KATP channel blockers, such as
glibenclamide and 5-hydroxydecanoic acid, diminish the
beneficial effects of short ischemic events on the cardiac tissue. The
original hypothesis to explain these observations involved plasma
membrane KATP channels. Recently, it has been
proven that the action of KCOs such as diazoxide concerns, in fact,
mitochondria and the mitoKATP channel (Garlid et
al., 1997
). In a complementary approach, it was shown that diazoxide
induced oxidation of mitochondrial flavoproteins, due to the activation
of mitoKATP channel, but did not activate plasma membrane KATP channels (Liu et al., 1998
). The
effects of diazoxide were completely and reversibly blocked by
5-hydroxydecanoic acid. Interestingly, exposure to
phorbol-12-myristate-13-acetate potentiated and accelerated the effect
of diazoxide (Sato et al., 1998
). These studies established that the
target for the protective effects of diazoxide in cardiac myocytes is
the mitoKATP channel rather than the plasma
membrane KATP channel (Fig.
5). Importantly, evidence for
mitoKATP channels as effectors of myocardial
preconditioning has also been demonstrated in human subjects (Ghosh et
al., 2000
).
|
The initial observations on the cardioprotective action of KCOs on
mitochondria were further confirmed and developed in a series of
reports. It has been shown that other KCOs such as pinacidil, cromakalim, and nicorandil modulate mitochondrial membrane potential, respiration, ATP generation, and mitochondrial
Ca2+ uptake (Holmuhamedov et al., 1998
; Sato et
al., 2000
). Other data suggest that activation and diazoxide-induced
translocation of the protein kinase C
-isoform to mitochondria
appears to be important for the protection mediated by the
mitoKATP channel (Wang et al., 1999
; Wang and
Ashraf, 1999
). Recently, it has been shown with the use of diazoxide
that ischemic preconditioning depends on the interaction between actin
cytoskeleton and mitochondria, and its protective action can be
abolished by disruption of the cytoskeleton by cytochalasin D (Baines
et al., 1999
). Interestingly, diazoxide was also effective in improving
the preservation of globally ischemic cold-stored hearts, as it occurs
during cardiac transplantation (Kevelaitis et al., 1999
, 2000
; Ahmet et
al., 2000
).
The main question remains how the opening of the
mitoKATP channel could protect cells against
ischemic injury. First, opening of the mitoKATP
channel followed by mitochondrial swelling could improve mitochondrial
ATP production and/or handling (Garlid, 2000
). In fact, diazoxide was
found to preserve mitochondrial function in ischemic rat heart. It has
been shown that hypoxia induces a decrease in the mitochondrial oxygen
consumption rate to approximately 40% of the prehypoxic value, and
treatment with diazoxide preserves the normal mitochondrial oxygen
consumption rate during hypoxia (Iwai et al., 2000
). Moreover, ATP
concentration was significantly increased in diazoxide-treated hearts
(Wang et al., 1999
). Second, the protective effect of
mitoKATP activation could be mediated by lowering
Ca2+ overloading of mitochondria (Holmuhamedov et
al., 1998
; Crestanello et al., 2000
). Third, it has been demonstrated
that opening of the mitoKATP channel may increase
ROS generation by mitochondria (Pain et al., 2000
). This increase could
lead to protein kinase C activation, which is known to be important
during cardioprotection. Additionally, the
mitoKATP channel seems to be involved in delayed preconditioning (Carroll and Yellon, 2000
), probably due to an altered
expression of "protective" proteins. It has been shown that
pretreatment of hippocampal neurons with KCOs cromakalim and diazoxide
increases the expression level of proteins involved in the control of
apoptosis, such as Bcl-2 and Bcl-XL (Jakob et al., 2000
). Moreover, inhibition of apoptosis induced by oxidative stress in cardiac cells was observed (Akao et al., 2001
). The presence
of a mitochondrial target for diazoxide in hippocampal mitochondria
recently has been observed (D
bska et al., 2001
).
| |
VI. Sulfonylureas and Mitochondria |
|---|
|
|
|---|
It is well known that antidiabetic sulfonylureas such as
glibenclamide (also known as glyburide) or glipizide bind to high affinity sulfonylurea receptors (SURs) in the plasma membrane of
various cell types (Ashcroft and Ashcroft, 1992
; Isomoto and Kurachi,
1997
). In pancreatic
-cells this causes a closure of the
ATP-sensitive K+ (KATP)
channel (Ashcroft and Ashcroft 1992
; Lazdunski, 1994
; Ashcroft, 2000
)
and initiates a chain of events that leads to the exocytotic release of
insulin (Miki et al., 1999
). The pancreatic
-cell SUR was cloned
(Aguilar-Bryan et al., 1995
) and identified as an element composing,
together with a K+ pore, the functional
KATP channel (Inagaki et al., 1995
). Similar channels are also present in the plasma membrane of smooth, skeletal, and cardiac muscle cells as well as in neurons (DeWeille, 1992
). The
channels are heterooctamers of four inwardly rectifying
K+ channels (Kir) and four SURs. Two members of
the Kir family, Kir6.1 and Kir6.2, appear capable of forming the pores
of KATP channels (Aguilar-Bryan et al., 1998
;
Babenko et al., 1998
; Seino, 1999
). These Kir subunits coassemble with
SURs encoded by either of two genes, SUR1 or
SUR2, to form functional KATP
channels. SURs belong to the superfamily of ATP-binding cassette
proteins characterized by the presence of two nucleotide-binding folds within the molecule. Moreover, it is likely that various SUR subunits in combination with Kir6.x subunits contribute to the functional diversity of KATP channels and determine various
pharmacological properties of these channels, including their
regulation by KCOs.
A potassium channel, with properties similar to those of the
KATP channel of the plasma membrane, was
described in the inner mitochondrial membrane and designated as the
mitoKATP channel (see Section
V.). This channel is also blocked by antidiabetic sulfonylureas (Inoue et al., 1991
; Paucek et al., 1992
). The
interaction of antitumor sulfonylureas with mitochondria has also been
described (Fig. 6) (Howbert et al.,
1990
).
|
A. Functional Effects of Antidiabetic Sulfonylureas on Mitochondria
The effects of antidiabetic sulfonylureas on mitochondrial
K+ transport have been observed both in intact
mitochondria (Szewczyk et al., 1997
) and in proteoliposomes
reconstituted with partly purified mitoKATP
channel (Paucek et al., 1992
). The mitoKATP channel became sensitive to glibenclamide only when opened by Mg2+, ATP, and physiological activators such as
GTP, or by the KCO diazoxide. In such an induced open state of the
mitoKATP channel, glibenclamide inhibited the
channel activity with a K1/2 of 1 to 6 µM (Jab
rek et al., 1998
).
Equilibrium binding studies performed with
[3H]glibenclamide reveal a single class of
low-affinity binding sites in intact rat liver mitochondria, with a
Kd of 4 µM (Szewczyk et al., 1996b
). In beef heart mitochondria the Kd for
glibenclamide binding is much lower, 300 nM (Szewczyk et al., 1997
).
Glibenclamide binding to mitochondria is modulated by SH reagents such
as N-ethylmaleimide and mersalyl (Szewczyk et al., 1999
).
It is important to mention that, due to the hydrophobicity of its
protonated form, glibenclamide is able to increase the proton conductance of the mitochondrial membrane (Szewczyk et al., 1997
). Additionally, antidiabetic sulfonylureas such as glibenclamide and
tolbutamide affect fatty acid oxidation due to the inhibition of
carnitine palmitoyltransferases (Patel, 1986
; Cook, 1987
) and also
block pyruvate carboxylase activity (White et al., 1988
).
B. Effect of Antitumor Sulfonylureas on Mitochondria
Diarylsulfonylureas are antitumor agents shown to have therapeutic
activity against both rodent solid tumors and xenografts of human
tumors in mice (Howbert et al., 1990
; Mohamadi et al., 1992
; Houghton
and Houghton, 1996
). Their mechanism of action is unknown but does not
appear to be the result of nonselective destruction of actively
dividing cell populations. In isolated liver mitochondria, both
N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea and
its N-4-methyl analog uncouple oxidative phosphorylation
(Thakar et al., 1991
). At concentrations below 50 µM, both compounds
exhibited a deleterious effect, causing damage to mitochondrial
functions. These data confirm that diarylsulfonylureas may lower
cellular ATP by uncoupling mitochondrial oxidative phosphorylation
(Thakar et al., 1991
).
Diarylsulfonylureas, such as
N-(4-chlorophenyl)aminocarbonyl-2,3-dihydro-1-indene-5-sulfonamide
(sulofenur) and
N-(4-chlorophenyl)aminocarbonyl-4-methylbenzene sulfonamide (LY181984), have also been shown to be effective antitumor agents (Fig. 6) (Stagg and Diasio, 1990
). Mitochondria have been shown
to accumulate sulofenur and therefore may be targets of drug action.
Many of the diarylsulfonylureas that were effective antitumor agents in
animal models were also uncouplers of mitochondrial oxidative
phosphorylation (Rush et al., 1992
). The mechanism of uncoupling
appeared to be related to a dissociable hydrogen ion, inasmuch as these
molecules had pKa values that ranged
from 6.0 to 6.2 and were hydrophobic. However, the mechanism of
antitumor activity does not appear to be the result of uncoupling,
because no correlation was evident between the inhibition of cell
growth and the uncoupling action of a variety of active and inactive antitumor diarylsulfonylureas.
| |
VII. The Mitochondrial Benzodiazepine Receptor |
|---|
|
|
|---|
Benzodiazepines are among the most widely prescribed drugs due to
their pharmacological actions in relieving anxiety, and as
anticonvulsants, muscle relaxants, or sedative hypnotics. These effects
are mediated in the central nervous system through postsynaptic plasma
membrane GABAA receptors that are
-aminobutyric acid-gated chloride channels. In addition to these
central-type benzodiazepine receptors, binding sites were also
identified in peripheral tissues, and this second class of sites was
termed the peripheral benzodiazepine receptor (PBR). The PBR first
characterized by Braestrup and coworkers (Braestrup and Squires, 1977
;
Braestrup et al., 1977
) is present in peripheral tissues such as
adrenal glands, kidney, and heart, as well as in the brain. The density
of the PBR is the highest in endocrine tissues such as adrenal gland,
testis, ovary, uterus, and placenta. PBR is also abundant in kidney,
heart, and platelets, but densities in these tissues are approximately
five times lower than that in adrenal gland. The PBR has been localized
in mitochondrial membranes, and nonmitochondrial localizations have
been observed in heart, liver, and testis. Additionally, the PBR was
found in mature erythrocytes, which lack mitochondria. The properties
and role of PBR were described in several review papers (McEnery, 1992
;
Parola et al., 1993
; Gavish et al., 1999
).
There is some terminological inconsistency concerning the mitochondrial
PBR. Some authors (e.g., Tatton and Olanow, 1999
) use this term to
describe an 18-kDa polypeptide with high affinity toward benzodiazepine
derivatives and isoquinoline carboxamides. This peptide has been
isolated from rat adrenal gland and characterized by
Antkiewicz-Michaluk et al., (1988a
,b
). However, other authors (e.g.,
McEnery et al., 1992
; Gavish et al., 1999
) consider the isoquinoline-binding protein (IBP) as one of at least three subunits of
the mitochondrial PBR. The other two components are the mitochondrial pore protein, porin, also known as voltage-dependent anion channel (VDAC), with a molecular mass of 32,000; and the adenine nucleotide translocase, with a molecular mass of 30,000. The reactivity of the
18-kDa polypetide toward benzodiazepines usually requires the
interaction of all three subunits.
IBP is a protein with five transmembrane domains usually associated
with the mitochondrial outer membrane. The cDNA for the 850-nucleotide
IBP mRNA has been cloned from a number of species. A detailed
description of the IBP gene was recently reviewed (Gavish et al.,
1999
).
The PBR complex is located in the contact sites between the outer and
the inner mitochondrial membranes. Its subunit composition roughly
coincides with that of the mitochondrial permeability transition pore
(Brustovetsky and Klingenberg, 1996
; Beutner et al., 1996
; Halestrap et
al., 1997b
) that opens under specific conditions and enables
unselective passage of molecules of up to 1.5 kDa between the
mitochondrial matrix and the cytoplasm (Bernardi et al., 1994
; Zoratti
and Szabó, 1995
). Recently, much attention has been directed
toward this pore, also called the "mitochondrial megachannel",
because of its postulated role in events leading to programmed cell
death (apoptosis) in multicellular organisms (Kroemer et al., 1997
,
1998
; Tatton and Olanow, 1999
). The role of the 18-kDa IBP in the
opening/closing transition of the pore and in its function in eliciting
apoptosis is not clear. On the other hand, other components, such as
cyclophillin D, have been described as obligatory functional elements
of the permeability transition pore (Halestrap et al., 1997a
) (for more
information see Section III. and Fig. 3).
Recently, using a cytoplasmic domain of IBP as a bait in the yeast
two-hybrid system, a new protein that specifically interacts with IBP
has been cloned (Gali
gue et al., 1999
). This protein, named
PRAX-1 (peripheral benzodiazepine receptor-associated protein 1),
exhibits several domains involved in protein-protein interactions such
as three proline-rich domains and three leucine-zipper motifs (Gali
gue et al., 1999
).
In contrast to the central benzodiazepine receptor, PBR
exhibits nanomolar affinity to benzodiazepine Ro5-4864
(4'-chlorodiazepam) and the isoquinoline carboxamide derivative
PK11195, and low affinity to benzodiazepine clonazepam.
Isoquinoline carboxamide derivatives, such as PK11195, bind
specifically to the 18-kDa IBP subunit, whereas PBR-specific
benzodiazepine ligands, such as Ro5-4864, bind to a site consisting of
porin, as well as adenine nucleotide translocase and the 18-kDa IBP
subunit (Garnier et al., 1994
). Other ligands for PBR, such as
2-aryl-3-indoleacetamide (Romeo et al., 1992
) and
N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide (Chaki et al., 1999
), were also described.
Benzodiazepine Ro5-4864 and isoquinoline carboxamide PK11195 are
the two most widely used PBR ligands. Based on the entropy-driven and
enthalpy-driven nature of ligand-receptor interactions, PK11195 has been classified as an antagonist and Ro5-4864 as an agonist
(Le Fur et al., 1983
). The functional significance of such
classification was recently confirmed in studies on the antiapoptotic activities of these PBR ligands (Bono et al., 1999
).
Protoporphyrin IX binds to PBR with nanomolar affinity and has been
suggested to be an endogenous ligand for PBR (Snyder et al., 1987
).
During heme biosynthesis, cytosolic coproporphyrinogen III traverses
the mitochondrial outer membrane and is converted via protoporphyrin IX
to heme, which is subsequently exported from mitochondria. This
suggests an involvement of PBR in mitochondrial heme synthesis (Verma
et al., 1987
; Woods and Williams, 1996
). Another candidate for
endogenous ligand for PBR is a 104-amino acid neuropeptide known as
diazapam binding inhibitor (Corda et al., 1984
). Also, a 16-kDa protein
called anthralin inhibits the specific binding of Ro5-4864 to PBR
(Mantione et al., 1988
).
PBR has been implicated in several mitochondrial functions, but its
exact physiological role is still unclear. In vitro studies using
isolated cells, mitochondria, and submitochondrial fractions demonstrated that PBR is present in steroid-synthesizing cells and is
involved in this kind of cell in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, known to
be the rate-determining step in steroid biosynthesis (Culty et al.,
1999
). The postulated role of PBR was recently reviewed in detail
(Gavish et al., 1999
). PBR agonist Ro5-4864 was found to strongly
protect against apoptosis induced by tumor necrosis factor-
in human
lymphoblastoid cell line U937 (Bono et al., 1999
). The potent
antiapoptotic effect might represent a major function for this receptor
as demonstrated by the lack of antiapoptotic activity of Ro5-4864 in
wild-type Jurkat cells (lacking the PBR receptor) and the reappearance
of this effect in PBR-transfected cells (Bono et al., 1999
).
Additionally, the blockade of the antiapoptotic effect of PBR agonist
by selective PBR antagonist PK11195 was observed (Bono et al., 1999
).
| |
VIII. Immunosuppressant Drugs and Mitochondria |
|---|
|
|
|---|
Cyclosporin A has potent immunosuppressive properties due to its
ability to block the transcription of cytokine genes in activated T
cells (Rovira et al., 2000
). It is well established that CsA forms a
complex with cyclophylin D, inhibiting the peptidyl-prolyl cis-trans isomerase activity of this protein. Additionally,
the CsA-cyclophilin complex inhibits the activity of calcineurin
(Rusnak and Mertz, 2000
). Calcineurin is a Ca2+-
and calmodulin-dependent serine/threonine phosphatase (protein phosphatase 2B) that regulates nuclear translocation and subsequent activation of the nuclear factor of activated T cells known as NFAT
transcription factor. Prevention of NFAT dephosphorylation, which is an
important step for its translocation to the nucleus, blocks cytokine
production. In addition to the calcineurin/NFAT pathway, recent studies
indicate that CsA also blocks the activation of stress-activated
protein kinase JNK (c-jun NH2-terminal kinase) and the mitogen-activated protein kinase p38 signaling pathway triggered by antigen recognition (for review see Matsuda and Koyasu, 2000
). Despite this beneficial role of CsA in organ transplantation, CsA has significant side effects such as hypertension, and renal and
muscle toxicity. Probably, these effects of CsA are related to ROS
generation (Buetler et al., 2000
).
CsA blocks the opening of the PTP (see Section
III.A.) because of its high affinity to cyclophilin D. This
effect is independent of the inhibition of calcineurin and the
immunosuppressive action of CsA, because the CsA analog
N-methyl-Val-4-cyclosporin lacks immunosuppressive
properties and is still able to block PTP (Zamzami et al., 1996
;
Hortelano et al., 1997
; Matsumoto et al., 1999
; Vergun et al., 1999
).
The blocking of PTP opening is most likely the mechanism underlying the
protective action of CsA against ischemic and ischemic/reperfusion
injuries. Such beneficial effects have recently been described in
ischemic and traumatic brain injury (TBI) in experimental animals
(Scheff and Sullivan, 1999
; Sullivan et al., 1999
, 2000
; Albensi et
al., 2000
; Li et al., 2000
). For example, animals subjected to
forebrain ischemia for 30 min exhibited extensive neuronal necrosis and
failed to survive, whereas injection of CsA (in combination with an
intracerebral lesion to open the blood-brain barrier) prolonged their
survival time, ameliorating brain damage and preventing secondary
mitochondrial dysfunction (Li et al., 2000
).
Although TBI often results in impaired learning and memory functions,
the underlying mechanisms are unknown, and there are currently no
treatments that can preserve such functions. Recently, the plasticity
at CA3-CA1 synapses in hippocampal slices from rats subjected to
controlled cortical impact TBI was studied (Albensi et al., 2000
).
Long-term potentia