Examining the Role of Mitochondria in Ca 2+ Signaling in Native Vascular Smooth Muscle JOHN G. McCARRON,* MARNIE L. OLSON,* CALUM WILSON,* † MAIRI E. SANDISON,* AND SUSAN CHALMERS* *Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK; † Department of Biomedical Engineering, University of Strathclyde, Glasgow, UK Address for correspondence: John G. McCarron, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, SIPBS Building, 161 Cathedral Street, Glasgow G4 0RE, UK. E-mail: john.mccarron@strath.ac.uk Received 15 October 2012; accepted 7 January 2013. ABSTRACT Mitochondrial Ca 2+ uptake contributes important feedback controls to limit the time course of Ca 2+ signals. Mitochondria regulate cytosolic [Ca 2+ ] over an exceptional breath of concentrations (~200 nM to >10 lM) to provide a wide dynamic range in the control of Ca 2+ signals. Ca 2+ uptake is achieved by passing the ion down the electrochemical gradient, across the inner mitochondria membrane, which itself arises from the export of protons. The proton export process is efficient and on average there are less than three protons free within the mitochondrial matrix. To study mitochondrial function, the most common approaches are to alter the proton gradient and to measure the electrochemical gradient. However, drugs which alter the mitochondrial proton gradient may have substantial off target effects that necessitate careful consideration when interpreting their effect on Ca 2+ signals. Measurement of the mitochondrial electrochemical gradient is most often performed using membrane potential sensitive fluorophores. However, the signals arising from these fluorophores have a complex relationship with the electrochemical gradient and are altered by changes in plasma membrane potential. Care is again needed in interpreting results. This review provides a brief description of some of the methods commonly used to alter and measure mitochondrial contribution to Ca 2+ signaling in native smooth muscle. Key words: Smooth muscle, mitochondria, calcium signalling, imaging Abbreviations used: [Ca 2+ ], cytoplasmic Ca 2+ concentration; AM, acetoxymethyl; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CCh, carbachol; DiOC6, 3,3-dihexyloxacarbocyanine iodide; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; IP 3 , inosi- tol 1,4,5-trisphosphate; IP 3 R, inositol 1,4,5-trisphosphate receptor; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocy- anine iodide; LetM1, leucine zippered-EF-hand containing trans- membrane protein 1; mAChR3, muscarinic type 3 receptor; MCU, mitochondrial uniporter; mNCX, mitochondrial Na + /Ca 2+ exchan- ger; PLC, phospholipase C; PTP, permeability transition pore; rhod- 2, rhodamine-like fluorophores; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; TMRE, tetramethylrhodamine ethyl ester; TMRM, tetramethylrhodamine methyl ester; DΨ M , mitochondrial membrane potential. Please cite this paper as: McCarron JG, Olson ML, Wilson C, Sandison ME, Chalmers S. Examining the role of mitochondria in Ca 2+ signaling in native vascular smooth muscle. Microcirculation 20: 317–329, 2013. FINE CONTROL OF Ca 2+ SIGNALING AND BIOLOGICAL RESPONSES Changes in the [Ca 2+ ] c trigger numerous vascular smooth muscle cell activities, which include cell division, growth, metabolism, contraction, and death. To enable the ion to modulate such a diversity of activities, there are a wealth of different types of Ca 2+ signals (the Ca 2+ toolkit, [6,45]) with various amplitudes, durations, and frequencies and the signal may be confined to particular parts of the cell. Each of these features (amplitude, frequency, duration, location) may selectively target Ca 2+ signals to particular physiolog- ical responses. A central requirement for the existence of the various temporal and spatial signals are local feedback processes which shape the Ca 2+ increase or restrict changes in the [Ca 2+ ] c to small parts of the cell. Mitochondria are of acknowledged significance in the feedback control of Ca 2+ signaling [15]. The organelle’s facility for rapid Ca 2+ uptake controls the Ca 2+ signal and the altered signal is transduced to a biological response by mitochondria themselves or by other parts of the cell. The two main sources of Ca 2+ are the extracellular fluid and the intracellular stores (the SR). Ca 2+ enters the cell from the extracellular fluid via channels on the plasma membrane such as the voltage-dependent and store- operated Ca 2+ channels. The other main Ca 2+ source is the SR DOI:10.1111/micc.12039 Invited Review ª 2013 John Wiley & Sons Ltd 317