Evidence for sigma-1-like receptors in isolated rat liver mitochondrial membranes

Abstract

1. Sigma (sigma) receptors have generated a great deal of interest on the basis of their possible roles in various pathologies, including cytoprotection. Although the exact function of sigma-1 (sigma(1)) receptors is not yet known, their role in the regulation of intracellular Ca(2+) levels and sterol biosynthesis, functions that could be assigned to mitochondria, are the only mechanisms described. 2. Using preparations of purified rat liver and brain mitochondria we demonstrate herein the presence of sigma-like binding sites. [(3)H](+)-pentazocine, a sigma(1) radioligand was used to label these sites. 3. In the liver, [(3)H](+)-pentazocine labelled one class of binding sites with high affinity (K(d)=3 nM), similar to that observed in liver microsomes and synaptic membranes. These sites were located on the outer mitochondrial membranes and displayed high affinity for other sigma(1) ligands namely, haloperidol, ifenprodil, carbetapentane or 1,3-di(2-tolyl)guanidine (DTG). 4. The presence of sigma(1) receptors on liver mitochondria was confirmed using double fluorescence immunostaining. 5. [(3)H](+)-pentazocine binding sites were also found on brain mitochondria but they appeared pharmacologically distinct to the liver ones as [(3)H](+)-pentazocine and typical sigma(1) ligands displayed lower affinities for these sites. Nevertheless, [(3)H](+)-pentazocine binding on both liver and brain mitochondria was modulated by progesterone, a putative endogenous ligand for sigma receptors. 6. Our data demonstrates the presence of [(3)H](+)-pentazocine binding sites with pharmacological characteristics identical to sigma(1) receptors on rat liver mitochondrial membranes. The pharmacological significance of these sites and their role on mitochondrial function remain unknown.

Figure 1

Association and dissociation kinetics of [³H](+)-pentazocine to rat liver mitochondria. Association experiment: Liver mitochondria (1 mg ml⁻¹) were incubated with 2 nM [³H](+)-pentazocine at 37°C. Specific binding in intact (B) and broken mitochondria (A). Dissociation experiment (C): after equilibrium was reached, dissociation was induced by dilution and specific binding monitored for 500 min (C). Data shown are plotted as fractions of the maximal binding value ([³H](+)-pentazocine bound=1) and are from typical experiments representing the means of duplicate determinations.

Figure 2

Representative equilibrium binding curve of [³H](+)-pentazocine binding to liver mitochondria. Concentration of [³H](+)-pentazocine (0.2 – 2 nM) was incubated with sonicated mitochondria (1 mg ml⁻¹) for 90 min at 37°C and non-specific binding was defined using 1 μM haloperidol. Equilibrium parameters were estimated by a non-linear regression analysis. In this particular experiment, K_d and B_max values were 2.78 nM and 1.20 pmol mg⁻¹ protein, respectively. (A) Direct plot. (B) Scatchard plot.

Figure 3

Effect of several σ receptor ligands on [³H](+)-pentazocine binding to rat liver mitochondria. Incubations were carried out with 2 – 3 nM [³H](+)-pentazocine at 37°C for 90 min. Data shown are from typical experiments and are plotted as fractions of the control binding value (in the absence of inhibitor: [³H](+)-pentazocine bound=1).

Figure 4

Relationship between the affinity of drugs for [³H](+)-pentazocine binding sites from liver mitochondria, liver microsomes and brain membranes. Plotted are pK_i values listed in Table 2. Regression line was obtained by least squares fit analysis of the data points.

Figure 5

[³H](+)-pentazocine binding sites are located on the outer mitochondrial membranes. Freshly isolated mitochondria (8 mg ml⁻¹) were treated or not with 0.6 mg digitonin mg⁻¹ protein in 1 ml Tris-sucrose buffer for 15 min at 4°C. After incubation the mitochondrial suspension was diluted and centrifuged at 15,000×g for 7 min. Cytochrome c oxidase activity, monoamine oxidase activity and [³H](+)-pentazocine (2 nM) binding were then assayed in the pellet. The maximal activities of the markers (100% values) were 1.57 nmol min⁻¹ mg⁻¹ protein for monoamine oxidase, 0.87 nmol min⁻¹ mg⁻¹ protein for cytochrome c oxidase, and 0.41 pmol mg⁻¹ protein for [³H](+)-pentazocine binding. Each data point is the mean±s.e.mean of duplicate measurements obtained from 3 – 4 experiments.

Figure 6

Colocalization of σ₁ receptor and mitochondrial Ab-1 marker in rat liver. As described under Methods rat liver sections were incubated with σ₁ receptor and mitochondria Ab-1 primary antibodies. Using conventional immunofluorescence the presence of σ₁ receptor (green) and mitochondria Ab-1 (red) in rat liver is clear. Confocal images of σ₁ receptor staining and mitochondria Ab-1 staining were merged, showing a colocalization (yellow) of their respective antigens. Magnification: ×20 (top) and ×40 (bottom).