Pflügers Arch - Eur J Physiol (2002) 443:754–761 DOI 10.1007/s00424-001-0774-9 Abstract Changes of cell volume and intracellular calci- um concentration ([Ca 2+ ] i ) in immortalized thick ascend- ing limb of Henle’s loop (TALH) cells were monitored using confocal laser scanning microscopy and fura-2 fluorescence, respectively. Reduction of the extracellular osmolarity from 600 to 300 mosmol/l induced cell swell- ing followed by regulatory volume decrease (RVD). Simultaneously, the [Ca 2+ ] i increased transiently. The calcium rise was not observed in calcium-free solution or in the presence of nifedipine, indicating that the change was, in the first place, due to the activation of a calcium influx. Application of ATP or caffeine in isotonic solu- tions increased transiently the [Ca 2+ ] i which revealed the existence of stores in TALH cells sensitive to inositol- 1,4,5 trisphosphate (IP 3 ) and ryanodine. To examine the possibility that the calcium influx might induce calcium release, manganese quenching experiments were per- formed. In hypotonic calcium-free solutions, the decay of the calcium-insensitive and calcium-sensitive fluores- cence occurred simultaneously. In the presence of extra- cellular calcium however, the calcium-sensitive wave- length revealed initial calcium influx followed by a cal- cium release from intracellular stores. Thus, the calcium influx was a prerequisite for the calcium release. We conclude that calcium-induced calcium release partici- pates in global calcium signalling during RVD of TALH cells. Keywords Calcium channel · Calcium signalling · Calcium stores · Osmoregulation · Regulatory volume decrease Introduction A reduction of extracellular osmolarity induces an in- crease in cell volume in most animal cells. Within min- utes of hypotonic stress, the cells reduce the swelling by a mechanism called regulatory volume decrease (RVD, for a recent review see [23]). Calcium has been found to play a role as a second messenger during cell volume regulation [25, 42]. In several cell types, RVD strictly depends on calcium influx. In these systems, the volume regulation can be inhibited by removing extracellular calcium or by the presence of calcium channel blockers [45, 47]. In other cell types, RVD is mainly dependent on calcium release from intracellular stores [2, 29, 38]. For example, in rat inner medullary collecting duct cells (IMCD), cell swelling in hyposmotic solutions leads to a biphasic calcium response, in which the early transient phase is due to the mobilization of calcium from intra- cellular stores while the later sustained phase represents entry of extracellular calcium across the plasma mem- brane [39, 40]. Moreover, the calcium release from intra- cellular stores is a necessary and sufficient condition for calcium entry as well for the RVD of IMCD cells. There is certain physiological similarity between the IMCD cells and cells from the thick ascending limb of Henley’s loop (TALH). Both IMCD cells and TALH cells are involved the regulation of urine osmolarity in the renal medulla. Water and urea permeability in both cell types is very low in the absence of antidiuretic hormone. IMCD cells as well as TALH cells use organic osmolytes to balance the high extracellular osmolarity in this part of the kidney [22]. In TALH cells calcium has been found to be a neces- sary factor for volume regulation [27]. A reduction of extracellular osmolarity from 315 to 180 mosmol/l induced a calcium signal that activated a K + channel in the luminal plasma membrane. Under this condition, the K + channel has been shown to be mainly responsible for the RVD. However, in the renal medulla interstitial con- centrations of osmotically active substances are generally much higher compared with those in other compartments H. Tinel ( ✉ ) Bayer AG, PH-R-CVII, PO Box 10 17 09, 42096 Wuppertal, Germany e-mail: hanna.tinel.ht@bayer-ag.de Tel.: +49-202-364040, Fax: +49-202-368009 H. Tinel · E. Kinne-Saffran · R.K.H. Kinne Max-Planck-Institut für molekulare Physiologie, Abteilung Epithelphysiologie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany ORIGINAL ARTICLE Hanna Tinel · Evamaria Kinne-Saffran Rolf K. H. Kinne Calcium-induced calcium release participates in cell volume regulation of rabbit TALH cells Received: 1 October 2001 / Revised: 26 November 2001 / Accepted: 26 November 2001 / Published online: 5 February 2002 © Springer-Verlag 2002