A Walk in the Memory, from the First Functional Approach up to Its Regulatory Role of Mitochondrial Bioenergetic Flow in Health and Disease: Focus on the Adenine Nucleotide Translocator

Thus, to find out whether ANT or ATP synthase determines the rate of ATP appearance outside mitochondria, this activity was measured in the presence of CAT and OLIGO. The former inhibits the transport but not the synthesis of ATP; on the contrary, the second inhibits synthesis without inhibiting the carrier.

By measuring the rate of the ATP efflux as a function of the concentration of each of the two inhibitors, the inhibition was immediate and increasing for the inhibitor of the reaction representing the limiting stage, i.e., CAT.

Although OLIGO inhibits ATP synthase, it is unable to inhibit the global process of ATP efflux until the rate of ATP synthesis via ATP synthase inhibited by OLIGO becomes lower than that of the carrier. Plotting the reciprocal of the speed measured as a function of the concentration of the non-penetrating inhibitor, the intercept of the straight line at the ordinate axis is a measure of the transport activity in the absence of an inhibitor, indicating that the ATP/ADP exchange determines the rate of ATP efflux. On the contrary, with OLIGO, not all points are at the same line: the y-axis intercept (equal to zero (OLIGO)) of the extrapolated lines, differed (1/V lower) from the activity measured at zero (OLIGO). Thus, we were able to conclude that the rate of ATP appearance outside mitochondria is governed by the rate of ADP/ATP exchange via ANT rather than the rate of ATP synthesis via ATP synthase.

This is one case in which (see above), as expected, the determination of the limiting stage could not be done through the use of a detergent since OXPHOS requires intact mitochondrial membranes. That is why, since ANT activity depends on the mitochondrial membrane potential mΔΨ, measurement of its activity was always preceded by that of the mΔΨ. The choice of substrates, the quality of mitochondria, and the buffer composition, to name a few, all affect mΔΨ, and as extension of this, the ADP–ATP exchange rate. It follows that another aspect to take into account when applying this method is the fact that, under the adopted experimental conditions, the energy for ATP synthesis was given by the oxidation of endogenous substrates. Sometimes, for experimental needs, to ascertain that mitochondria themselves can drive ATP synthesis without any limitation due to energy deficit, we energized mitochondria present in cell homogenates by adding β-hydroxybutyrate (5 mM), a respiratory substrate that enters mitochondria via diffusion with an increase in membrane potential. If the rate of ATP production was not significantly increased under these conditions, we concluded that the mitochondria themselves could generate the electrochemical proton gradient needed for ATP synthesis. Conversely, prevention of ΔμH+ formation from endogenous substrates by the addition of a cocktail of the electron flow inhibitors rotenone, antimycin A, mixothiazole, and cyanide resulted in blocking ATP production.

Furthermore, extreme care was taken to use enough HK/G6PDH-coupled enzymes to ensure a non-limiting ADP-regenerating system for the measurement of ATP production. Therefore, as controls, it was always verified that (i) the coupled enzymatic system used to detect ATP outside mitochondria was not rate limiting per se by showing that the addition of further ATP resulted in an increase in the rate of absorbance, (ii) whereas no rate increase occurred following the addition of both substrate and enzyme components of the ATP D.S.

Before going into the specific topic whose pivot is the impairment of ANT in different pathological conditions, we want to trace in a few lines the path carried out by this protein in the last 30 years.

Surely an important milestone was achieved in 1996, when it was discovered that ANT, along with other proteins, also functions as a non-specific pore upon stimulation by ions and a variety of molecules, such as calcium (Ca2+) and ROS, as shown by the reconstitution of native purified ANT into liposomes [106]. A second important breakthrough highlighted the critical involvement of ANT as a major player in the execution of the mitochondrial apoptosis pathway in various pathophysiological and experimental models [56,107], according to which ANT defined cell fate by cooperating with pro-apoptotic proteins such as Bax, viral proteins (for example, viral protein R (Vpr) from HIV-1, PB1-F2 from influenza), or by participating in the mitochondrial polyprotein complex in diverse model systems such as human carcinoma cell lines, cardiomyocytes, and neurons [56,108]. More recently, the fact that each ANT isoform has a specific role in influencing cell fate, notably in cancer cells [56,109], represented another flag raised by the research on ANT.

From this roundup of milestones it can be deduced that ANT is a bi-functional protein, and that it harbors two opposite functions, both of which are intricately involved in the control and regulation of cell fate [56]: a vital function important for the maintenance of mitochondrial function to ensure the life of the cell, and another lethal one, leading the cell to death.

It follows that it is intuitive to think that impairing its function leads to severe diseases.

At this point in our study, beyond briefly reviewing human diseases associated with mutations or altered expression of ANT, we will focus on some disease states in which ANT function is compromised. In addition, we will describe some particular conditions—either pathological or conditions in which the effect on ANT by an effector with which mitochondria or cells are treated is observed—in which ANT was in the spotlight, thus leading to monitoring its dysfunction by applying the method of the ATP detecting system (ATP D.S.).

ANT1, one of the four isoforms (see Section 3.5), has been so far the only to directly cause mitochondrial diseases.

Based on investigations of mitochondrial dysfunction it is evident that aberrant ANT1 activity causes human disease, certainly as a result of diminished activity—as in autosomal dominant progressive external ophthalmoplegia (adPEO) and Senger’s syndrome—and possibly also due to its overexpression in facioscapulohumeral muscular dystrophy (FSHD) [110].

However, the discovery that ANT1 dysfunction can cause adPEO not only revealed a surprising role in mtDNA stabilization, but also provided one example of an mtDNA deletion disorder caused by an autosomal gene defect.

In Senger’s syndrome, an autosomal recessive disease characterized by hypertrophic cardiomyopathy, mitochondrial myopathy, lactic acidosis, and congenital cataracts [111], it appears likely that ANT1 depletion is secondary to a mutation in a gene that functions in processing the translocase. No mutations have been found in ANT1, causing speculation that the transcription, translation, or posttranslational modification of ANT1 may be affected [112]. In fact, this is so: the loss of AGK, the mitochondrial acylglycerol kinase involved in lipid metabolism, may result in a decrease in ANT by affecting its biogenesis.

In contrast to ANT1 deficiency, overexpressed ANT1 may contribute to the pathogenesis of other diseases such as FSHD, characterized by the cumulative progression of muscle weakness in the face, feet, shoulders, and hips, along with occasionally sensorineural hearing loss [113]. FSHD is a highly variable autosomal dominant neuromuscular disorder. Although overexpression of FRG1, a gene involved in pre-mRNA splicing, was proposed as being responsible for FSHD, other studies proposed the overexpression of ANT1 and increased oxidative stress in FSHD muscles [110].

At this point, we would like to briefly describe one of the latest studies performed by Kliment et al. [114], just to underline how research on ANT is still fervent 30 years after its discovery and also to introduce the ANT2 isoform, which is involved in the development of chronic obstructive pulmonary disease (COPD). The authors observed that ANT2 expression is reduced in lung tissue from patients affected by COPD, and in a mouse smoking model. ANT1 and ANT2 overexpression resulted in enhanced oxidative respiration and ATP flux, stimulating airway surface liquid hydration by ATP and maintaining ciliary beating after cigarette smoke exposure, which are key functions of the airway. Therefore, the canonical protein, i.e., ANT (with ANT1, 2, and 3 present in human lungs), not only regulates mitochondrial metabolism but also plays a central function in airway epithelial biology, notably airway hydration, which promotes ciliary function.

Unlike some more specialized reviews on the “ADP/ATP carrier” topic, the purpose of this review section is to focus only on the analyses of the functional activity of ANT, allowing us to assess its role in most varied pathophysiological contexts. Therefore, we selectively report here only those studies in which mitochondria, isolated from tissues/cells or present in the homogenates, are respiring and phosphorylating, where ANT works in own environment, the only condition that allows us to assess its role in any pathophysiological context that mimics the real situation in which the cell finds itself.

We are of the opinion that two general approaches can be used to investigate mitochondrial bioenergetics. The first approach requires the isolation of mitochondria from cells or tissues of different origins to design experimental conditions and protocols aimed at ensuring their integrity and mirroring the functional activity—as accurately as possible—of the environment of the intact cell. The great temptation is to modify the experimental conditions in order to amplify the phenomenon to be studied.

Instead, the second approach is to work with intact cells, where the gain of physiological relevance is balanced by the greater complexity of the preparation and the difficulty of accessing the mitochondria in situ. As we will see later, the research in our group focused on the development of techniques to improve access to the bioenergetics of mitochondria in situ by miniaturizing certain experimental approaches that are normally used to carry out measurements on isolated mitochondria.

With the intention of making a real reconnaissance of studies in which ANT appears, we will start with 1988, the historic year in which the ATP detecting system was developed and tested, and—through various types of studies regarding the functioning of ANT in various conditions ranging from apoptosis to necrosis (as regards neurons) to the condition in which there is an explosion of oxidative stress, or even the condition in which laser light or a photosensitization condition with specific drugs manifest their effect on the translocator, and again to ANT as a target of the two Alzheimer’s proteins and so on—we will get to the present day (Figure 2).

We will look at them one by one. For each of them—we would like to reiterate—we resorted to the procedure that allows for the continuous monitoring of ATP efflux from mitochondria incubated with ADP, using the ATP detecting system.

As mentioned earlier, the reason why in 1988 we wanted to develop a measurement of the ADP/ATP carrier was the evidence accumulated in previous years that mitochondria were targets of light irradiation (for ref. see [115,116,117]).

Passarella et al. [116,117] proved that irradiation of isolated RLM with a helium neon laser causes both an increase in the electrochemical proton gradient and ATP extra-synthesis. Given that cytosolic ATP availability could be dependent on the rate of ADP/ATP exchange, investigation into the possible effects of He-Ne laser irradiation on the activity of the ADP/ATP translocator seemed worthwhile. Therefore, in this first study, the development of such a method was presented as well as its first application to investigate the ADP/ATP antiport in mitochondria irradiated by a He-Ne laser. Irradiation of isolated RLM was also found to increase the rate of ADP/ATP exchange [104], likely due to the increase in the electrochemical proton gradient that occurs owing to the irradiation of mitochondria.

Some years later, after a large body of work had been accumulated showing that He-Ne lasers stimulate both biogenesis and bioenergetics of mitochondria, specifically determining an increase in mitochondrial ATP synthesis, we made a correct observation: endogenous adenine nucleotides, substrates involved in ATP synthesis, are also themselves irradiated under conditions in which ATP extra-synthesis occurred, that is, when the mitochondrion was irradiated, as in [104]. Therefore, the question that arose was intriguing: could a He-Ne laser light affect the biochemical features of AMP, ADP, and ATP? It had already been observed that NADH was sensitive to ruby and He-Ne laser irradiation and that irradiation of substrates and enzymes with non-coherent light proved to change their biochemistry [118]. Therefore, adenine nucleotides irradiated with 3 Joules/cm2 fluence (10 mW/cm2 fluence rate) showed altered biochemical behaviors when used as substrates for certain mitochondrial reactions in isolated RLM. It was observed that only certain enzymes are able to recognize irradiated adenine nucleotides, and that this property is not a substrate property, but rather concerns the substrate–enzyme complex [118]. We arrived at this conclusion since different effects were observed for the same irradiated substrate in different reactions. As a result of irradiation, ADP affinity for the ADP/ATP carrier increased, whereas it decreased when the ADK reaction was studied. No difference between irradiated or non-irradiated ADP was found in the ATP synthase reaction.

In addition, mitochondria were the target of photosensitization, a reaction to light mediated by a light-absorbing molecule that causes biochemical changes, ultimately leading to cell death [115,119]. Photodynamic therapy, which precisely consists of treatment with a hematoporphyrin derivative followed by photoradiation with visible light, was a promising approach to the treatment of various cancers. The first step in the photoinduced process is light absorption; then the photosensitizer, excited by light, is converted to a triplet excited state; this latter species leads to the formation of singlet oxygen and radicals that oxidize biomolecules. If, on the one hand, hematoporphyrin derivatives were, at that time, the most commonly used drugs for fluorescent visualization and photosensitization of various tumors [120,121], and in particular photofrin II (PF II) was indicated as the most active component by [120], on the other hand, mitochondria were indicated as the primary target of PF II photodamage [121]. In support of this, a depletion of the intracellular ATP pool was found in cancer cells in tissue culture or in situ, possibly due to the impaired mitochondrial function owing to PF II-induced photosensitization, which likely produces ROS, mainly singlet oxygen, that damages cellular components, leading to cytotoxicity [122].

It was a duty and of undoubted interest to clarify the mechanism of interaction between mitochondria and hematoporphyrin. Therefore, as part of the Researchers’ Exchange Agreement between the National Research Council (CNR, Italy) and the Institut National de la Santè e de la Recherche Medicale (INSERM, France), this topic—project research title: “The effect of hematoporphyrin photosensitization on mitochondrial and cellular activities”—was the object of study by A.A., in quality by Chercheur Associè, during her stay in Paris. Although the results obtained proved to be extremely interesting, now we leave them aside to focus on the question of photosensitization action on ANT. Strong non-competitive inhibition of the ADP/ATP exchange carrier was observed in isolated RLM when challenged with hematoporphyrin-sensitized photodynamic action, showing that the adenine nucleotide carrier is a major target of photodynamic action, which causes OXPHOS impairment [123]. In order to understand why PF II photosensitization was able to cause impairment of ANT activity, in a dedicated study, Atlante et al. [124] observed that photodynamic action exerted by PF II on ANT, a carrier protein sensitive to mercurial compounds able to bind to mitochondrial thiols—the target of singlet oxygen [125]—was prevented if mitochondria were treated with mersalyl, thus suggesting that SH groups present in carrier molecules are the functional groups affected by PF II photosensitization.

Within the therapy of some pathologies involving mitochondria, structurally different pharmacological agents, including antitumors, immunosuppressants, anesthetics, etc, have been designed to specifically affect mitochondrial functions. In other cases, the same drugs (such as anti-viral drugs) with primary targets in other cellular locations were found to impair mitochondrial functions as side effects.

Really interesting is the study conducted on 3-azido-3′-deoxythymidine (AZT), the most widely used antiretroviral drug in acquired immunodeficiency syndrome (AIDS) therapy, by D.V. during her PhD [126,127]. It seemed interesting to us to propose it in this context because this study is an interesting example in which, although the AZT molecule competitively inhibits ANT, it is not taken up into the mitochondria through it. ATP D.S. as a method to follow ADP/ATP exchange is well suited in this situation to better highlight ADP/ATP translocator impairment by AZT as one of the biochemical processes responsible for ATP deficiency syndrome, the main severe side effect associated with antiviral drug therapy.

First, the authors showed that AZT per se can enter mitochondria, as shown by both isotopic and HPLC measurements, and, more, that its transport occurs in a carrier-mediated manner. Furthermore, although externally added AZT was found to inhibit the ANT-mediated ADP/ATP exchange in both mitochondria and mitoplasts, the drug does not use the ADP/ATP translocator to enter the organelles. This conclusion is based both on the failure of AZT to cause an efflux of intramitochondrial ATP, and more importantly, on the insensitivity of AZT uptake to CAT, a powerful inhibitor of the ADP/ATP carrier.

The impairment of the ADP/ATP translocator could well be the biochemical process that exerts a direct effect on OXPHOS, which in turn could be responsible for the ATP deficiency syndrome induced in cells treated with AZT and which is proposed to be a severe AZT side effect in AIDS therapy [128,129]. If AZT uptake in mitochondria and AZT inhibition of ADP/ATP transport play a significant role in ATP deficiency syndrome, then the discovery of a proper inhibitor of AZT transport in mitochondria, as well as a proper AZT chemical modification that could prevent ADP/ATP carrier impairment and/or its uptake into mitochondria, could be valuable goals for further research.