Differential responses to histamine and cyclo-pentyladenosine in subclones of the DDT1MF-2 cell line.

The hamster vas deferens derived smooth muscle cell line, DDTIMF-2, has been shown to possess functional histamine HI-receptors, stimulation of which leads to the production of the second messenger inositol 1,4,5trisphosphate (IP,) and subsequent release of calcium from intracellular stores [1,2]. This cell line also possesses both adenosine A,and A,-receptors which are coupled respectively negatively and positively to adenylate cyclase [3,4]. We have shown previously that stimulation of the A,-receptor can also lead to inositol phospholipid hydrolysis in this cell line [5]. In this study we have isolated and maintained a total of eight clonal lines (TW1-8) from a population of DDT,MF-2 cells which we believe to be heterogenous. The clonal cell lines were established using a combination of microscopy and single cell isolation techniques. To this end, a suspension of the DDT,MF-2 cell line was diluted to give a theoretical cell density of 1 ce11/2pl. 2p1 aliquots of this resulting suspension were examined to determine the presence of a single cell before transfer to a well of a 96 well cluster dish. The clonal lines were maintained under conditions previously described €or the parent line [5] i.e. at 37T in a humidified air/CO, (9O:lO) environment. Cells were initially maintained in conditioned Dulbecco's modified Eagle's medium (DMEM) supplemented with foetal calf serum (10% v/v) and 2mM L-glutamine in 75cm2 flasks. When the cells lines had become established basic supplemented DMEM was used instead of conditioned medium. Cells for assay were grown in 24 well cluster dishes. The clonal cells were fed after 48h and were passaged every 5 days (1:s split ratio). Confluent monolayers of clonal cells were Joaded with ['HI-inositol for 24h prior to challenge with a range of agonists. Following assay, the accumulation of total [ -inositol phosphates in the disrupted cell monolayer was determined using anion exchange chromatography and liquid scintillation counting. The relative sizes of the responses to stimulation by histamine (HA) and N6cyclopentyladenosine (CPA) varied markedly between the clonal lines (see Fig.1). For example the responses to CPA (1OOnM) were 2.0