Biochimice et Biophysica Acta, 1010(1989)265-269 Elsevier 265 BBA 12411 Dual elevation of cycfic AMP and ]nositol phosphates in response to mechanical deformation of rnur/ne osteoblasts Jonathan R. Sandy 1, Sajeda Meghji 1, Richard W. Farndale 3 and Murray C. Meikle 2,4 i Oral Surgery Research Laboratories and 2 Department of Orthodontics, Eastman Dental Hospital and Institute of Dental Surgery. London, "~ Department of Biochemistry, University of Cambridge, Cambridge and 4 Strangeways Research Laboratory, Cambridge (U. K.) (Received 5 July 1988) (Revisedmanuscript received22 September1988) Key words: Mechanical stress; Inositolphosphate;cyclicAMP; (Mouseosteoblast) Mechanica| de|ormation of bone ceils was thought to be mediated via prostagland|n production and the cyclic AMP pathway. We present evidence that the pbospho|nosRide pathway is also activated by mechanical stress. We find that |nositol phosphate production, hut not glycerophosphoinositol production, is elevated, and the activation of adenylate cyclase is relatively small These results are not compatih|e with the proposal that mechanical deformation of bone ce||s acts solely via prost~glandin synthesis. Introduction It has long been recognised that mechanical forces regulate bone mass [1], yet the mechanism by which deformation of bone is translated into cellular responses remains unclear. The importance of mechanical stimula- tion of bone is emphasised by the loss of bone mass which occurs in disuse osteoporosis [2]. The cyclic nucleotide, cyclic AMP (cAMP) has been implicated [3-6] following prostaglandin production from per- turbed bone cell membranes [7]. Additionally, changes in Ca 2+ and K + fluxes at the cell membrane may result from mechanical stimulation applied to tissues [8-10]. Distortion of cytoskeletal elements may also play a role in this transduction [11]. Recently, other second messengers have been de- scribed, including the eicosanoids and the phospholipid metabolites, inositol phosphates (InsPs) and di- acylglycerol [12-15]. The Ca 2+ changes previously re- ported in mechanically deformed osteoblasts [8] might have occurred as a cAMP-dependent Ca 2+ flux through cell membranes [16], but since we have recently shown that prostaglandins also activate phospholipid hydroly- Abbreviations: lnsPs, inositol phosphates; PTH, parathyroid hormone; PGE z, prostaglandin E2; GroPlns, glycerophosphoinosi- tol; DMEM, Dulbecco's modified Eagle'smedium;IBMX,3-isobutyl- 1-methylxanthine. Correspondence: J.R. Sandy, Oral Surgery Research Laboratories, Eastman Dental Hospitaland Instituteof Dental Surgery,256 Grays Inn Road, LondonWC1X8LD, U.K. sis, it is unlikely that the effects of mechanical stress will be restricted to cAMP elevation alone [17]. Hydrolysis of phosphatidylinositol 4,5-bisphosphate in response to hormones binding to cell surface recep- tors leads to inositol trisphosphate, Ins(1,4,5)P 3, forma- tion and the release of calcium from intracellular stores [18,19]. Further phosphorylation of Ins(1,4,5)P3 yields Ins(1,3,4,5)P4 which may control calcium entry at the plasma membrane [20]. We have recently demonstrated_ in osteoblasts that parathyroid hormone (PTH) caused an increase in inositol trisphosphate (InsP3) accumula- tion, whereas prostaglandin E2 (PGE2) caused an equal elevation of InsPs and g!ycero!pho~phoinesite! (GroPIns), as well as cAMP. Furthermore, indometha- cin abolished the action of PTH on inositol phosphate mobilisation suggesting that prostaglandins mediate the effects of PTH on inositol phosphate production [17]. If mechanical stress also acts on the osteoblasts by pros- taglandin production, as previously reported [5], it is likely that inositol phosphates as well as cAMP will be elevated. This hypothesis is the basis of this communi- cation. Materials and Methods Osteoblast preparation Osteoblasts were extracted from the calvariae of 5- day-old mice by sequential enzyme digestion using a modification of an established method [21]. Calvariae were dissected out and washed in Hank's buffered salt solution (Gibco, Paisley, U.K.). The initial digest was for 15 min using 0.25% trypsin (Gibco) and 0167-4889/89/$03.50 © 1989ElsevierSciencePublishersB.V.(Biomedical Division)