Amiloride derivatives induce apoptosis by depleting ER Ca2+ stores in vascular endothelial cells : Amiloride derivatives induce ER Ca2+ depletion

Park, KS,Poburko, D,Wollheim, CB,Demaurex, N Wiley (Blackwell Publishing) 2009 British journal of pharmacology Vol.156 No.8

<P>BACKGROUND AND PURPOSE: Amiloride derivatives are blockers of the Na(+)/H(+) exchanger (NHE) and at micromolar concentrations have protective effects on cardiac and brain ischaemia/reperfusion injury but at higher concentrations also induce apoptosis. Here, we aimed to elucidate the mechanism related to this cytotoxic action. EXPERIMENTAL APPROACH: We quantified the expression of genes associated with endoplasmic reticulum (ER) stress and measured changes in luminal ER Ca(2+) concentration ([Ca(2+)](ER)) with a 'cameleon' indicator, D1ER. KEY RESULTS: Amiloride derivatives induced apoptosis in vascular endothelial cells, an effect that increased at alkaline extracellular pH. The potency order for cytotoxicity was 5-(N,N-hexamethylene)-amiloride (HMA) > 5-(N-methyl-N-isobutyl) amiloride > 5-(N-ethyl-N-isopropyl) amiloride (EIPA) >> amiloride. HMA dose-dependently increased the transcription of the ER stress genes GADD153 and GADD34 and rapidly depleted [Ca(2+)](ER), mimicking the effects of the sarco/endoplasmic reticulum ATPase (SERCA) inhibitor thapsigargin. The NHE1-specific inhibitor HOE 694 inhibited NHE activity by 87% but did not alter [Ca(2+)](ER). The decrease in [Ca(2+)](ER) evoked by amiloride derivatives was also observed in HeLa cells and was mirrored by an increase in cytosolic Ca(2+) concentration. CONCLUSIONS AND IMPLICATIONS: Amiloride derivatives disrupt ER and cytosolic Ca(2+) homeostasis by a mechanism unrelated to NHE inhibition, most likely by interfering with the activity of SERCA. We propose that ER Ca(2+) depletion and subsequent ER stress provide a rationale framework for the apoptotic effects of amiloride derivatives.</P>

Defective Mitochondrial Function and Motility Due to Mitofusin 1 Overexpression in Insulin Secreting Cells

Park, Kyu-Sang,Wiederkehr, Andreas,Wollheim, Claes B. The Korean Society of Pharmacology 2012 The Korean Journal of Physiology & Pharmacology Vol.16 No.1

Mitochondrial dynamics and distribution is critical for their role in bioenergetics and cell survival. We investigated the consequence of altered fission/fusion on mitochondrial function and motility in INS-1E rat clonal ${\beta}$-cells. Adenoviruses were used to induce doxycycline-dependent expression of wild type (WT-Mfn1) or a dominant negative mitofusin 1 mutant (DN-Mfn1). Mitochondrial morphology and motility were analyzed by monitoring mitochondrially-targeted red fluorescent protein. Adenovirus-driven overexpression of WT-Mfn1 elicited severe aggregation of mitochondria, preventing them from reaching peripheral near plasma membrane areas of the cell. Overexpression of DN-Mfn1 resulted in fragmented mitochondria with widespread cytosolic distribution. WT-Mfn1 overexpression impaired mitochondrial function as glucose- and oligomycin-induced mitochondrial hyperpolarization were markedly reduced. Viability of the INS-1E cells, however, was not affected. Mitochondrial motility was significantly reduced in WT-Mfn1 overexpressing cells. Conversely, fragmented mitochondria in DN-Mfn1 overexpressing cells showed more vigorous movement than mitochondria in control cells. Movement of these mitochondria was also less microtubule-dependent. These results suggest that Mfn1-induced hyperfusion leads to mitochondrial dysfunction and hypomotility, which may explain impaired metabolism-secretion coupling in insulin-releasing cells overexpressing Mfn1.

Defective Mitochondrial Function and Motility Due to Mitofusin 1 Overexpression in Insulin Secreting Cells

박규상,Andreas Wiederkehr,Claes B. Wollheim 대한약리학회 2012 The Korean Journal of Physiology & Pharmacology Vol.16 No.1

Mitochondrial dynamics and distribution is critical for their role in bioenergetics and cell survival. We investigated the consequence of altered fission/fusion on mitochondrial function and motility in INS-1E rat clonal β-cells. Adenoviruses were used to induce doxycycline-dependent expression of wild type (WT-Mfn1) or a dominant negative mitofusin 1 mutant (DN-Mfn1). Mitochondrial morphology and motility were analyzed by monitoring mitochondrially-targeted red fluorescent protein. Adenovirus- driven overexpression of WT-Mfn1 elicited severe aggregation of mitochondria, preventing them from reaching peripheral near plasma membrane areas of the cell. Overexpression of DN-Mfn1 resulted in fragmented mitochondria with widespread cytosolic distribution. WT-Mfn1 overexpression impaired mitochondrial function as glucose- and oligomycin-induced mitochondrial hyperpolarization were markedly reduced. Viability of the INS-1E cells, however, was not affected. Mitochondrial motility was significantly reduced in WT-Mfn1 overexpressing cells. Conversely, fragmented mitochondria in DN- Mfn1 overexpressing cells showed more vigorous movement than mitochondria in control cells. Movement of these mitochondria was also less microtubule-dependent. These results suggest that Mfn1-induced hyperfusion leads to mitochondrial dysfunction and hypomotility, which may explain impaired metabolism-secretion coupling in insulin-releasing cells overexpressing Mfn1.

Mitochondrial phosphate transport during nutrient stimulation of INS-1E insulinoma cells

Quan, X.,Das, R.,Xu, S.,Cline, G.W.,Wiederkehr, A.,Wollheim, C.B.,Park, K.S. North-Holland 2013 Molecular and cellular endocrinology Vol.381 No.1

Here, we have investigated the role of inorganic phosphate (P<SUB>i</SUB>) transport in mitochondria of rat clonal β-cells. In α-toxin-permeabilized INS-1E cells, succinate and glycerol-3-phosphate increased mitochondrial ATP release which depends on exogenous ADP and P<SUB>i</SUB>. In the presence of substrates, addition of P<SUB>i</SUB> caused mitochondrial matrix acidification and hyperpolarisation which promoted ATP export. Dissipation of the mitochondrial pH gradient or pharmacological inhibition of P<SUB>i</SUB> transport blocked the effects of P<SUB>i</SUB> on electrochemical gradient and ATP export. Knock-down of the phosphate transporter P<SUB>i</SUB>C, however, neither prevented P<SUB>i</SUB>-induced mitochondrial activation nor glucose-induced insulin secretion. Using <SUP>31</SUP>P NMR we observed reduction of P<SUB>i</SUB> pools during nutrient stimulation of INS-1E cells. Interestingly, P<SUB>i</SUB> loss was less pronounced in mitochondria than in the cytosol. We conclude that matrix alkalinisation is necessary to maintain a mitochondrial P<SUB>i</SUB> pool, at levels sufficient to stimulate energy metabolism in insulin-secreting cells beyond its role as a substrate for ATP synthesis.

Oxidative stress and calcium dysregulation by palmitate in type 2 diabetes

Luong Dai Ly,Shanhua Xu,최성경,하채명,THOUDAMTHEMIS,차승규,Andreas Wiederkehr,Claes B. Wollheim,이인규,박규상 생화학분자생물학회 2017 Experimental and molecular medicine Vol.49 No.-

Free fatty acids (FFAs) are important substrates for mitochondrial oxidative metabolism and ATP synthesis but also cause serious stress to various tissues, contributing to the development of metabolic diseases. CD36 is a major mediator of cellular FFA uptake. Inside the cell, saturated FFAs are able to induce the production of cytosolic and mitochondrial reactive oxygen species (ROS), which can be prevented by co-exposure to unsaturated FFAs. There are close connections between oxidative stress and organellar Ca2+ homeostasis. Highly oxidative conditions induced by palmitate trigger aberrant endoplasmic reticulum (ER) Ca2+ release and thereby deplete ER Ca2+ stores. The resulting ER Ca2+ deficiency impairs chaperones of the protein folding machinery, leading to the accumulation of misfolded proteins. This ER stress may further aggravate oxidative stress by augmenting ER ROS production. Secondary to ER Ca2+ release, cytosolic and mitochondrial matrix Ca2+ concentrations can also be altered. In addition, plasmalemmal ion channels operated by ER Ca2+ depletion mediate persistent Ca2+ influx, further impairing cytosolic and mitochondrial Ca2+ homeostasis. Mitochondrial Ca2+ overload causes superoxide production and functional impairment, culminating in apoptosis. This vicious cycle of lipotoxicity occurs in multiple tissues, resulting in β-cell failure and insulin resistance in target tissues, and further aggravates diabetic complications.

Essential Role of Mitochondrial Ca²⁺ Uniporter in the Generation of Mitochondrial pH Gradient and Metabolism-Secretion Coupling in Insulin-releasing Cells

Quan, Xianglan,Nguyen, Tuyet Thi,Choi, Seong-Kyung,Xu, Shanhua,Das, Ranjan,Cha, Seung-Kuy,Kim, Nari,Han, Jin,Wiederkehr, Andreas,Wollheim, Claes B.,Park, Kyu-Sang American Society for Biochemistry and Molecular Bi 2015 The Journal of biological chemistry Vol.290 No.7

<P>In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca<SUP>2+</SUP> influx, which triggers insulin exocytosis. The mitochondrial Ca<SUP>2+</SUP> uniporter (MCU) mediates Ca<SUP>2+</SUP> uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca<SUP>2+</SUP> rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca<SUP>2+</SUP>. Suppression of the putative Ca<SUP>2+</SUP>/H<SUP>+</SUP> antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca<SUP>2+</SUP>-induced matrix acidification. These results demonstrate that MCU-mediated Ca<SUP>2+</SUP> uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells.</P>