Role of mitochondria in Ca 2 oscillations and shape of Ca 2 signals in pancreatic acinar cells C. Camello-Almaraz, G.M. Salido, J.A. Pariente, P.J. Camello * Department of Physiology, Faculty of Veterinary Sciences, University of Extremadura, 10071 Ca Âceres, Spain Received 12 June 2001; accepted 25 September 2001 Abstract We studied the role of mitochondria in Ca 2 signals in fura-2 loaded exocrine pancreatic acinar cells. Mitochondrial depolarization in response to carbonylcyanide-p-try¯uoromethoxyphenyl hydrazone or rotenone assessed by confocal microscopy using rhodamine-123) induced a partial but statistically signi®cant reduction in the decay of Ca 2 signals under different experimental conditions. Spreading of Ca 2 waves evoked by the pancreatic secretagogue cholecystokinin cholecystokinin octapeptide was accelerated by mitochondrial inhibitors, whereas the cytosolic Ca 2 concentration [Ca 2 ] i ) oscillations in response to physiological levels of this hormone were suppressed by rotenone and carbonylcyanide-p-try¯uoromethoxyphenyl hydrazone. Oligomycin, an inhibitor of mitochondrial ATP synthase, did no affect either propagation of calcium waves nor [Ca 2 ] i oscillations. Individual mitochondria of rhod-2 loaded acinar cells showed heterogeneous matrix Ca 2 concentration increases in response to oscillatory and maximal levels of cholecystokinin octapeptide. On the other hand, using Ba 2 for unequivocal study of capacitative calcium entry we found that mitochondrial inhibitors did not affect this process. Our results show that although the role of mitochondria as a Ca 2 clearing system in exocrine cells is quantitatively secondary, they play an essential role in the spatial propagation of Ca 2 waves and in the development of [Ca 2 ] i oscillations. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Ca 2 oscillations; Mitochondria; Capacitative Ca 2 in¯ux; Pancreas 1. Introduction Changes in cytosolic-free calcium concentration are an ubiquitous messenger system involved in the regulation of a plethora of cellular processes, from contraction or secre- tion to differentiation or apoptosis. This factor is exqui- sitely controlled by two types of transport systems. On one hand, different types of channels allow diffusion of Ca 2 ions into the cytosol from the intracellular calcium stores inositol 1,4,5-trisphosphate IP3) and ryanodine recep- tors) or from the extracellular medium Ca 2 in¯ux chan- nels).Ontheotherhand,Ca 2 is actively removed from the cytosol towards the internal pools by sarco-endoplasmic reticulum Ca 2 ATPase SERCA)) or to the extracellular space via plasma membrane Ca 2 ATPase PMCA) and Na  /Ca 2 exchange). The fact that mitochondria accumulate Ca 2 , described more than 20 years ago in liver [1], and since then in other tissues [2], for a review see [3]), prompted the concept that mitochondriamightshapeCa 2 signals inmammalian cells. In the last few years, new techniques have allowed both determination of mitochondrial matrix Ca 2 concentration and a detailed evaluation of perturbations of Ca 2 signals in response to experimental impairment of mitochondrial function. These techniques have con®rmed in situ the pre- viously assumed role of mitochondria as a low af®nity Ca 2 buffer, showing that these organelles take up Ca 2 during cytosolic Ca 2 signals [4±7], for reviews see [3,8]). An immediateconsequenceofmitochondrialCa 2 importisthe control of mitochondrial metabolic function [7,9]. The other consequence of this activity is that mitochon- dria contribute to shape [Ca 2 ] i signals. First, mitochon- dria contribute to the decay phase of [Ca 2 ] i signals, an aspect studied in numerous tissues [10±14]. Second, this uptake activity has an impact on spatial propagation of signals, although there are con¯icting reports ranging from Biochemical Pharmacology 63 2002) 283±292 0006-2952/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII:S0006-295201)00830-9 * Corresponding author. Tel.: 34-927-25-71-54;fax: 34-927-25-71-54. E-mail address: pcamello@unex.es P.J. Camello). Abbreviations: [Ca 2 ] i , cytosolic Ca 2 concentration; [Ca 2 ] m , mito- chondrial Ca 2 concentration; CCE, capacitative calcium entry; CCK, cholecystokinin octapeptide; IP3, inositol 1,4,5-trisphosphate; FCCP, carbonylcyanide-p-tryfluoromethoxyphenyl hydrazone; PMCA, plasma membrane Ca 2 ATPase; C m , mitochondrial membrane potential; SERCA, sarco-endoplasmic reticulum Ca 2 ATPase.