Natural diterpenoid; ADP/ATP

ATP, the principal energy currency of the cell, fuels most biosynthetic reactions in the cytoplasm by its hydrolysis into ADP and inorganic phosphate. Because resynthesis of ATP occurs in the mitochondrial matrix, ATP is exported into the cytoplasm while ADP is imported into the matrix. The exchange is accomplished by a single protein, the ADP/ATP carrier. Here we have solved the bovine carrier structure at a resolution of 2.2 A by X-ray crystallography in complex with an inhibitor, carboxyatractyloside. Six alpha-helices form a compact transmembrane domain, which, at the surface towards the space between inner and outer mitochondrial membranes, reveals a deep depression. At its bottom, a hexapeptide carrying the signature of nucleotide carriers (RRRMMM) is located. Our structure, together with earlier biochemical results, suggests that transport substrates bind to the bottom of the cavity and that translocation results from a transient transition from a 'pit' to a 'channel' conformation. [1]

---

The experiments presented in Fig. 1 were carried out with freshly prepared mitochondria. The results shown in Panel A, B, and C, illustrate the effect of carboxyatractyloside (CAT) and cyclosporin A (CSA) on mitochondrial Ca2+ efflux, mitochondrial swelling, and transmembrane potential. As seen in Panel A, trace a, the addition of 2 µM CAT initiates a fast discharge of accumulated matrix Ca2+. Trace b indicates that CSA inhibited CAT-induced Ca2+release. Trace a in Panel B shows swelling of mitochondria incubated in the basic medium that contained additionally 50 µM CaCl2. In trace b, it can be seen that CAT addition induced a large amplitude swelling. Trace c shows that this reaction was inhibited by CSA. Panel C depicts the influence of CAT and CSA on the transmembrane electric gradient. Trace a shows that the addition of CAT induced a fast collapse of the transmembrane potential. Trace b indicates that the drop in transmembrane potential (Δψ) was inhibited by CSA. We are aware that the above described results have been previously reported (see Zoratti et al., 2005, for a review). However, we considered it necessary to perform these experiments to be able to take these results as reference to those carried out in 24 h-aged mitochondria, which are described in the following.[2]

Male rats (10 rats/group) were treated with phenobarbital (PB), phenylbutazone (PBZ), stanozolol (3 inducers of cytochrome P450-dependent enzymes), piperonyl butoxide (PBO; a P450 inhibitor), cobaltous chloride (CoCl2; an inhibitor of hemoprotein synthesis), 5,6-benzoflavone (BNF; an inducer of cytochrome P448 dependent enzymes), cysteine [CYS; a glutathione (GSH) precursor], or ethyl maleate (EM; a GSH depletor). The rats were then given a calculated LD50 dosage (13.5 mg/kg of body weight) of carboxyatractyloside (CAT) intraperitoneally. Clinical signs of toxicosis, duration of illness, lethality, gross lesions, and hepatic and renal histopathologic lesions were recorded. Seemingly, (i) CAT toxicosis has independent lethal and cytotoxic components (PBZ decreased lethality and cytotoxicity; CoCl2 decreased cytotoxicity but not lethality; BNF decreased duration of illness, and perhaps lethality, but not cytotoxicity); (ii) CAT cytotoxicity could be partly due to an active metabolite formed by de novo-synthesized, P450-/P448-independent hemoprotein (PBZ and CoCl2 had anticytotoxic effects, but PB, stanozolol, PBO, and BNF did not); (iii) CAT detoxification may occur partly through a hemoprotein-independent, PBZ-inducible enzyme, and partly through a P448-dependent (BNF-inducible) enzyme; and (iv) CAT detoxification apparently is not P450 or GSH-dependent because PB, stanozolol, and CYS had no beneficial effects, and PBO, CoCl2, and EM did not enhance toxicosis. Metabolism of CAT may have a role in its cytotoxic and lethal effects. [4]

Mitochondrial preparation [2]
Liver mitochondria were isolated from Wistar rats after homogenization of the tissue in 0.25 M sucrose-1 mM EDTA, adjusted to pH 7.3, and following the standard centrifugation procedure. Mitochondrial ageing was attained after storing the preparation, 26 mg protein per mL, during 24 h at 4 °C. Protein was measured by the Lowry method (Lowry et al., 1951).

---

Calcium movements [2]
Calcium uptake and release was followed in a double beam spectrophotometer at 675–685 nm using 50 µM of the indicator Arsenazo III, by incubating 2 mg of mitochondrial protein in 3 mL of a basic medium containing 125 mM KCl; 10 mM succinate; 3 mM phosphate; 10 mM HEPES; 50 µM CaCl2 was added, the medium was adjusted to pH 7.3 with Tris-base. In addition, the medium contained 100 µM ADP, 2 µg oligomycin, and 5 µg rotenone.

---

Mitochondrial swelling [2]
Changes in mitochondrial volume were estimated at 540 nm, by incubating 2 mg protein from mitochondria in 3 mL of the basic medium. Arsenazo III was not added.

---

Transmembrane potential [2]
Transmembrane electric gradient was determined incubating 2 mg mitochondrial protein in 3 mL of the basic medium and followed at 511–533 nm by using 10 µM of the hydrophobic cation dye Safranine, instead of Arsenazo III.

---

ADP exchange reaction [2]
In order to analyze back exchange of the nucleotide, 10 mg protein from freshly prepared and aged mitochondria were preloaded with ADP by incubating in the basic medium supplemented with 1 mM [3H]-ADP (sp. act. 1500 cpm/nmol). After 20 min incubation, the mixture was centrifuged at 12,000 rpm during 10 min and the pellet was washed once; the final pellet was suspended in 0.25 mM sucrose–Tris, pH 7.3. Then, 1 mg ADP-loaded mitochondria was incubated 30 s in 1 mL of the basic medium containing additionally 60 µM ADP. After incubation, 0.2 mL was withdrawn and filtered through a 0.45-µm pore size filter. The radioactivity contained in mitochondria and retained in the filter was measured in a scintillation counter.

---

Cytochrome c determination [2]
Cytochrome c content was assayed according to Correa et al. (2007). Briefly, 2 mg mitochondrial protein was added to 3 mL of the basic mixture, containing in addition 100 µM ADP, 5 µg rotenone, 2 µg oligomycin, and 50 µM CaCl2; after 5 min incubation, the samples were centrifuged 10 min at 18,000 g, the supernatants were precipitated with thrichloroacetic acid, and the pellets were washed once. Protein (50 µg) was loaded onto 15% acrylamide SDS-PAGE gels, and transferred to a PVDF membrane for immunodetection. Cytochrome c content in mitochondria was evaluated using a primary monoclonal antibody against cytochrome c (1:1000 dilution) and an alkaline-phosphate-conjugated secondary antibody.

---

Aconitase activity [2]
The activity of this enzyme was measured according to Hausladen and Fridovich (1994). In short, mitochondrial protein was solubilized by adding 0.05% Triton X-100 containing 25 mM phosphate, pH 7.2, followed by the addition of 0.6 mM magnesium sulfate, 1 mM citrate, and 0.1 mM NADP. The formed cis-aconitate was measured spectrophotometrically at 240 nm.

---

Analysis of mitochondrial DNA [2]
Mitochondrial DNA disruption was assayed essentially as described (García and Chávez, 2007). Briefly, DNA was extracted from mitochondria suspended in 100 mM NaCl, 40 mM Tris, 20 mM EDTA, pH 7.8, and 1% SDS with the addition of 0.2 mg RNAase A, and incubated for 30 min at 37 °C. After incubation at 37 °C, proteins were precipitated and the DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1, vol/vol). The genetic material was analyzed in agarose gel and visualized with ethidium bromide. Other experimental conditions were as indicated in the respective legends of the figures.

[1]. Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside. Nature. 2003 Nov 6;426(6962):39-44.

[2]. Cyclosporin A is unable to inhibit carboxyatractyloside-induced permeability transition in aged mitochondria. Comp Biochem Physiol C Toxicol Pharmacol. 2009 Apr;149(3):374-81.

[3]. Effects of carboxyatractyloside a structural analogue of atractyloside on mitochondrial oxidative phosphorylation. Life Sci II. 1971 Sep 8;10(17):961-8..

[4].Toxicologic study of carboxyatractyloside (active principle in cocklebur--Xanthium strumarium) in rats treated with enzyme inducers and inhibitors and glutathione precursor and depletor. Am J Vet Res. 1982 Jan;43(1):111-6.

Carboxyatractyloside is a diterpene glycoside.
Carboxyatractyloside has been reported in Coffea with data available.

---

Free fatty acids are closely involved in the permeability transition process by locking the adenine nucleotide translocase in the cystosol side (Schönfeld and Bohnensack, 1997). As shown, this process was completely inhibited by CSA, as demonstrated by the fact that, in the presence of the immunosuppressant, aged mitochondria retained Ca2+ and were able to build up an electric transmembrane gradient. However, the finding that, notwithstanding the presence of CSA, the addition of low concentrations of carboxyatractyloside/CAT brought about opening of the non-specific pore was quite unexpected. The protective action of CSA has been attributed to its interaction with the peptidylprolyl cis-trans isomerase cyclophilin D (CyP) (Woodfield et al., 1998). Apparently, such a reaction occurred in CSA-exposed aged mitochondria, since these organelles behaved as freshly prepared mitochondria. A simple explanation would be that the content of CyP in aged mitochondria might be lower than in freshly prepared. However, as indicated, the amount of this enzyme in both, aged and fresh mitochondria, was quite similar. The reason for CAT-induced permeability transition turning CSA-insensitive seems to be in agreement with the findings by Bodrova et al. (2000). These authors demonstrated that the addition of carboxyatractyloside/CAT after myristate induces CSA-sensitive uncoupling; however, when CAT was added after myristate it produced a CSA-insensitive drop in Δψ. Their explanation is that ANT participates in the CSA-sensitive uncoupling when being part of the non-specific pore complex, but the ANT that does not take part in the pore complex formation is involved in the CSA-insensitive permeability transition. Another finding in this work was the release of cytochrome c from aged mitochondria. Cytochrome c release to the cytosol triggers formation of the apoptosome, resulting in activation of caspases (Borutaite and Brown, 2008); thus, the release of cytochrome c would explain the cellular apoptosis that occurs in aged tissue (Hoye et al., 2008). Finally, it is tempting to propose that, being permeability transition a common substrate of degenerative diseases, the immunosuppressant cyclosporin A would be a useful pharmacological tool for their treatment. In fact, studies by Borlongan et al. (2002) demonstrate that CSA is neuroprotective in animal models of Parkinson and Huntington diseases. [2]

C31H44O18S2-2.2[K+]

846.996860000001

846.124

C, 43.96; H, 5.24; K, 9.23; O, 34.00; S, 7.57

33286-30-5

Carboxyatractyloside tripotassium;77228-71-8; Carboxyatractyloside dipotassium;33286-30-5; 35988-42-2 (free acid);

20055804

White to off-white solid powder

2.598

6

18

13

51

1600

12

CC(C)CC(=O)O[C@@H]1[C@H]([C@@H]([C@@H](CO)O[C@H]1O[C@H]2C[C@@]3(C)[C@@H]4CC[C@H]5C[C@]4(CC[C@@H]3C(C2)(C(=O)[O-])C(=O)[O-])[C@H](C5=C)O)OS(=O)(=O)O)OS(=O)(=O)O.[K+].[K+]

AQFATIOBERWBDY-LNQSNDDKSA-N

InChI=1S/C31H46O18S2/c1-14(2)9-21(33)47-24-23(49-51(42,43)44)22(48-50(39,40)41)18(13-32)46-26(24)45-17-11-29(4)19-6-5-16-10-30(19,25(34)15(16)3)8-7-20(29)31(12-17,27(35)36)28(37)38/h14,16-20,22-26,32,34H,3,5-13H2,1-2,4H3,(H,35,36)(H,37,38)(H,39,40,41)(H,42,43,44)/t16-,17+,18-,19+,20+,22-,23+,24-,25+,26-,29+,30-/m1/s1

(1R,4S,7S,9S,10S,13R,15S)-15-hydroxy-7-[(2R,3R,4R,5R,6R)-6-(hydroxymethyl)-3-(3-methylbutanoyloxy)-4,5-disulfooxyoxan-2-yl]oxy-9-methyl-14-methylidenetetracyclo[11.2.1.01,10.04,9]hexadecane-5,5-dicarboxylic acid

Carboxyatractyloside; 33286-30-5; (1R,4S,7S,9S,10S,13R,15S)-15-hydroxy-7-[(2R,3R,4R,5R,6R)-6-(hydroxymethyl)-3-(3-methylbutanoyloxy)-4,5-disulfooxyoxan-2-yl]oxy-9-methyl-14-methylidenetetracyclo[11.2.1.01,10.04,9]hexadecane-5,5-dicarboxylic acid; SNP1XL23E6; PH9ATM6F5U; (1R,4S,7S,9S,10S,13R,15S)-15-hydroxy-7-{[(2R,3R,4R,5R,6R)-6-(hydroxymethyl)-3-[(3-methylbutanoyl)oxy]-4,5-bis(sulfooxy)oxan-2-yl]oxy}-9-methyl-14-methylidenetetracyclo[11.2.1.0^{1,10}.0^{4,9}]hexadecane-5,5-dicarboxylic acid; kaur-16-ene-18,19-dioic acid, 15-hydroxy-2-((2-o-(3-methyl-1-oxobutyl)-3,4-di-o-sulfo-beta-D-glucopyranosyl)oxy)-, dipotassium salt, (2beta,15alpha)-; (1R,4R,7R,9R,10R,13R,15S)-7-((2S,3R,4R,5S,6S)-6-(hydroperoxymethyl)-3-(3-methylbutanoyloxy)-4,5-disulfooxyoxan-2-yl)oxy-15-hydroxy-9-methyl-14-methylidenetetracyclo(11.2.1.01,10.04,9)hexadecane-5,5-dicarboxylic acid;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~100 mg/mL (~118.06 mM)
DMSO : ~25 mg/mL (~29.52 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (1.48 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 1.25 mg/mL (1.48 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 1.25 mg/mL (1.48 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 100 mg/mL (118.06 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

 (Please use freshly prepared in vivo formulations for optimal results.)