An implication of novel methodology to study pancreatic acinar mitochondria under in situ conditions

Abstract

Mitochondria maintain numerous energy-consuming processes in pancreatic acinar cells, yet characteristics of pancreatic mitochondrial oxidative phosphorylation in native conditions are poorly studied. Besides, it is not known which type of solution is most adequate to preserve functions of pancreatic mitochondria in situ. Here we propose a novel experimental protocol suitable for in situ analysis of pancreatic mitochondria metabolic states. Isolated rat pancreatic acini were permeabilized with low doses of digitonin. Different metabolic states of mitochondria were examined in KCl- and sucrose-based solutions using Clark oxygen electrode. Respiration of digitonin-treated, unlike of intact, acini was substantially intensified by succinate or mixture of pyruvate plus malate. Substrate-stimulated respiration rate did not depend on solution composition. In sucrose-based solution, oligomycin inhibited State 3 respiration at succinate oxidation by 65.4% and at pyruvate plus malate oxidation by 60.2%, whereas in KCl-based solution, by 32.0% and 36.1%, respectively. Apparent respiratory control indices were considerably higher in sucrose-based solution. Rotenone or thenoyltrifluoroacetone severely inhibited respiration, stimulated by pyruvate plus malate or succinate, respectively. This revealed low levels of non-mitochondrial oxygen consumption of permeabilized acinar cells. These results suggest a stronger coupling between respiration and oxidative phosphorylation in sucrose-based solution.

High‐throughput respiration screening of single mitochondrial substrates using permeabilized CHO cells highlights control of mitochondria metabolism

“…The OUR and stimulating effects of ADP determined in OUR study were in a similar range with literature data of permeabilized CHO cells , other mammalian cells , or isolated mitochondria from different tissues . However, an absolute comparison between different studies is difficult since the respiration rates are specific for substrates or substrate combinations, tissue, species, and cell line and also dependent on experimental conditions and history of cells, for example the training state and fitness level of cells .…”

Mechanisms of respiration intensification of rat pancreatic acini upon carbachol‐induced Ca2+ release

“…This allowed studying respiration at oxidation of exogenous substrates, namely pyruvate, malate and succinate, which do not stimulate respiration of intact pancreatic acini (Manko et al . ). As established by previous studies, the mitochondria of permeabilized cells are functionally active (Manko et al .…”

“…As established by previous studies, the mitochondria of permeabilized cells are functionally active (Manko et al . ). In addition, rhodamine 123 fluorescence proved the capability of permeabilized cell's mitochondria to maintain membrane potential (Fig.…”

“…), but 95–100% of acinar pancreacytes are trypan positive (Manko et al . ). Succinate (5 m m ) or mixture of pyruvate (5 m m ) and malate (5 m m ) were used as oxidative substrates.…”

Experimental substantiation of permeabilized hepatocytes model for investigation of mitochondria in situ respiration

“…This method is applied in various degrees to skeletal muscle fibers [5], smooth muscle cells [6], hepatocytes [7], pancreacytes [8] and enable to maintain the interaction between mitochondrial respiration and other cell processes. The advantage of using permeabilized cells is that, unlike the isolated mitochondrial fraction, entire cell population of mitochondria is studied in its natural environment [9].…”