Bone Vol. 19, No. 2 August 1996:101-106 ELSEVIER Expression of Voltage-Operated Ca 2+ Channels in Rat Bone Marrow Stromal Cells In Vitro M. R. PRESTON, A. J. EL HAJ, and S. J. PUBLICOVER School of Biological Sciences, University of Birmingham, Edgbaston, UK The expression of voltage-operated Ca 2÷ currents (VOCCs) in bone marrow stromal cells cultured for 3-30 days has been studied by the use of the whole-cell patch-clamp technique. Both low-voltage-activated (LVA) and high-voltage-activated (HVA) VOCCs were recorded. LVA currents were first de- tectable after 6-7 days in culture and reached a peak of expression at 8 days, after which both the amplitude and frequency of expression of the current fell rapidly. The cur- rent was virtually undetectable in cells cultured for more than 15 days. The HVA current was detectable after 3 days in culture and reached a peak of both amplitude and fre- quency of expression after 1-2 weeks. This current was ex- pressed consistently throughout the remaining culture pe- riod. In cultures treated with dexamethasone (10 -s mol/L) peak expression of LVA currents still occurred at 7-8 days, but currents were enhanced approximately threefold. Ex- pression of LVA currents was maintained to the end of the culture period. Expression of HVA currents was not signifi- cantly modified by treatment of cultures with dexametha- sone. Examination of the biophysical and pharmacological (blockade by Ni 2+ and diphenylhydantoin) properties of the LVA current in these cells suggests that they may have simi- larities with the LVA T currents of neuronal cells. (Bone 19:101-106; 1996) Key Words: Rat; Bone; Marrow stromal cells; Calcium chan- nels; T current; L current. Introduction Patch clamp studies have demonstrated the presence of voltage operated Ca 2+ currents (VOCCs) in a variety of cultured bone- derived cells and cell lines. ROS 17/2.8 osteosarcoma cells ex- press a high voltage activated (HVA) VOCC, 6"9 the MC3T3-EI cell line expresses a low-voltage activated (LVA), transient VOCC, 1 and both the UMR 106 line and osteoblast-like cells derived from rat neonate calvarium have been reported to express both of these types of current. 3'4'10'14 Rat bone marrow stromal cells maintained in culture for periods of 10-30 days express primarily a HVA current. LVA currents in these cells are small in comparison to HVA currents and are expressed in less than 15% of records. However, treatment with the corticosteroid Address for correspondence and reprints: Dr. S. J. Publicover, School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. dexamethasone greatly increases expression. 16 The HVA cur- rents found in these cells are dihydropyridine sensitive, and are probably of the L type, but the LVA channels, though considered to be possibly of the T type, have not been characterized. 16 In neuronal cell cultures LVA currents are often the first type of voltage-operated cation channel to be expressed, 5,ts'2~ some- times appearing within a few hours of plating out. However, HVA VOCCs (e.g., L-type currents) are subsequently expressed and become the major contributors to total voltage sensitive Ca 2+ current. 5 It has been suggested that this sequence of events may be related to cell differentiation, both in neurons and other ex- citable cells, depolarizations produced by T-current activation being of significance in development. 7'2° A similar sequence of events has recently been described in calvarial-derived cells. 12 LVA current is expressed in these cells after 3 days in culture, the HVA current appearing 4-5 days later. Subsequently the ratio of HVA:LVA current increases. 2 In the light of these data we have further examined the expression of VOCCs in marrow stromal cells cultured with and without dexamethasone for 2-30 days. In this communication we briefly describe further character- ization of the LVA current observed in marrow stromal cells and we report a more extensive investigation of the relationship be- tween time in culture, the presence of dexamethasone and current expression. It is shown that in control cultures a window of LVA current expression occurs at 7-14 days. In dexamethasone treated cultures LVA currents are detected earlier and the level of ex- pression and persistence of LVA currents are significantly enhanced. Methods Tissue Culture Bone marrow cells from 120 g Wistar rats were prepared after the method of Maniatopoulos et al. 13 Femora were dissected aseptically and washed four times in alpha-MEM (minimal es- sential medium) containing 5x the culture concentration of anti- biotics (see below). After removal of epiphyses, the marrow was flushed with culture medium injected through a 21-gauge needle. Single cells were obtained by repeated passage through a 19- gauge needle and filtration through sterile 100 ~m 2 mesh. Cells were seeded at a density of approximately 3×104 cells/mL onto 35 mm plastic dishes (Falcon and Bibby) and maintained at 37°C in a humidified atmosphere consisting of 95% air/5% CO 2. Cul- ture medium was alpha-MEM with 15% (v/v) fetal bovine serum, ascorbate (50 Ixg/mL), B glycerophosphate (10 mmol/L), peni- © 1996 by Elsevier Science Inc. 101 8756-3282/96/$15.00 All rights reserved. Pll $8756-3282(96)00136-6