Abstract

Rat neurotensin (NT) receptor (NTR) cDNA was subcloned into the pRC-CMV expression vector and transfected into 293 cells, and cellular clones that stably expressed the NTR were isolated and characterized. [3H]NT binding to membranes prepared from the NTR cDNA-transfected cells displayed specificity and saturability, with an apparent Kd of 1.25 nM and a Bmax of 43.4 pmol/mg of protein (approximately 3.5 x 10(6) binding sites/cell). NT stimulated an increase in [3H]inositol phosphate levels in the NTR-expressing cells up to 2500% of basal levels. The response was time and dose dependent, with an EC50 of 10.4 nM. NT also stimulated cAMP formation in these cells, with an EC50 of 27.0 nM. In addition, NT evoked an increase in the level of intracellular calcium. Approximately 60% of the calcium rise was attributable to the release of intracellular stores and 40% was attributable to calcium influx. Although NTR occupancy has been shown to stimulate cGMP formation in several brain preparations and cell lines, NT was unable to mediate cGMP synthesis in the NTR-expressing 293 cells. We found that 293 cells have guanylate cyclase activity but have undetectable levels of nitric oxide synthase (NOS) activity. Because it was possible that the production of nitric oxide is required as the mediator of NT-induced cGMP synthesis, we subcloned NOS cDNA into the pCEP4 expression vector and transiently expressed it in the NTR cells. We report that NT increased cGMP levels up to 375% of basal levels when NOS cDNA was coexpressed and that the increase was completely inhibited by the NOS inhibitor N omega-nitro-L-arginine. NT-induced cGMP accumulation was time and dose dependent, with an EC50 of 1.7 nM. To our knowledge, this is the first report of NT mediating cGMP formation with a cloned receptor and the first evidence that NT-induced cGMP accumulation requires the production of nitric oxide.