Identification by flow cytometry of two distinct rhodamine‐123‐stained mitochondrial populations in rat liver

Abstract

Isolated rat liver mitochondria were split into three fractions of increasing density when applied to a Percoll gradient. NADH-ubiquinone oxidoreductase, succinate dehydrogenase and cytochrome-c oxidase but not F,-ATPase activities increased with density as well as respiratory rate in state 3 and the respiratory control index. Flow cytometry of mitochondrial density fractions stained with rhodaminerevealed the occurrence in each density fraction of two distinct mitochondrial populations with different fluorescence intensity. The high fluorescence population was minor and its proportion decreased with density. The extent of high fluorescence population staining depended on the deenergized state of the mitochondria suggesting that this population represents an immature form of the mitochondria which may develop into a fully functional organelle by the incorporation of structural and/or functional proteins.

Postnatal changes in rhodamine‐123 stained mitochondrial populations are sensitive to protein synthesis inhibitors but mimicked in vitro by atp

“…Under the same circumstances, the percentages of mitochondria fluorescence populations changed markedly during the first hour of extrauterine life (Table 2). Thus, the percentage of the HFP sharply decreased while that of the LFP increased ( Table 2), suggesting that the HFP is an immature form of mitochondria that is converted into the mature one, the LFP [7,8], which is the major mitochondrial form in the adult rat liver [6]. In addition, the postnatal increase in the LPF/HFP ratio observed in our experiments (Table 2) may be related to the postnatal improvement in mitochondrial function (Table 1) Pollak et al [1,17] postulated that the acquisition of energy-transducing mitochondria after birth would be mediated by mitochondrial enrichment in ATP.…”

“…These results suggest that the conversion of the HFP into the LFP depends on the synthesis of mitochondrial proteins coded either by nuclear or by mitochondrial DNA. Since the changes in LFP/HFP ratio are probably associated with rh-123 binding to the F,F,-ATPase complex [6,7,11,13], it may be suggested that proteins involved in LFP-HFP interconversion are subunits of this enzyme. This is in agreement with our previous suggestion that the HFP is transformed into the LFP by the incorporation of some components of F,F,-ATPase, resulting in an increase in mitochondrial efficiency [7].…”

“…Mitochondria were stained by incubation with rh-123 (10 &ml) at 37°C for 15 min in the dark. The unbound dye was removed by centrifugation and the pellet washed twice with isolation medium [6]. All measurements of mitochondrial fluorescence and 90" angle light scatter (SSC) were made for at least 10,000 events/test with the FACStar flow cytometer using the Consort 30 software program (Bectotiickinson, San Jose, Ca, USA).…”

“…Likewise, flow cytometry analysis of isolated liver mitochondria stained with rh-123 has revealed the occurrence of two mitochondrial populations with different intensities of fluorescence, i.e. a high fluorescence population (HFP) and a low fluorescence population (LFP) [6]. The percentages of these populations change as postnatal development progresses and with the increase in mitochondrial respiratory efficiency [7,8].…”

N‐acetylcysteine impairs survival of luteal cells through mitochondrial dysfunction

“…In turn, R 123 binds also to sites freely accessible in deenergized mitochondria (60). Therefore, these sites may contribute to the fluorescence structure arising from the oxidation of DHR to R 123.…”