THE CLONED NEUROTENSIN RECEPTOR MEDIATES CYCLIC-GMP FORMATION WHEN COEXPRESSED WITH NITRIC-OXIDE SYNTHASE CDNA

Rat neurotensin (NT) receptor (NTR) cDNA was subcloned into the pRC-CM V expression vector and transfected into 293 cells, and cellular clone s that stably expressed the NTR were isolated and characterized. [H-3] NT binding to membranes prepared from the NTR cDNA-transfected cells d isplayed specificity and saturability, with an apparent K-d of 1.25 nM and a B-max of 43.4 pmol/ mg of protein (similar to 3.5 x 10(6) bindi ng sites/cell). NT stimulated an increase in [H-3]inositol phosphate l evels in the NTR-expressing cells up to 2500% of basal levels. The res ponse was time and dose dependent, with an EC(50) of 10.4 nM NT also s timulated cAMP formation in these cells, with an EC(50) 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 releas e of intracellular stores and 40% was attributable to calcium influx. Although NTR occupancy has been shown to stimulate cGMP formation in s everal brain preparations and cell lines, NT was unable to mediate cGM P synthesis in the NTR-expressing 293 cells. We found that 293 cells h ave guanylate cyclase activity but have undetectable levels of nitric oxide synthase (NOS) activity. Because it was possible that the produc tion of nitric oxide is required as the mediator of NT-induced cGMP sy nthesis, we subcloned NOS cDNA into the pCEP4 expression vector and tr ansiently expressed it in the NTR cells. We report that NT increased c GMP levels up to 375% of basal levels when NOS cDNA was coexpressed an d that the increase was completely inhibited by the NOS inhibitor N-om ega-nitro-L-arginine. NT-induced cGMP accumulation was time and dose d ependent, with an EC(50) of 1.7 nM. To our knowledge, this is the firs t report of NT mediating cGMP formation with a cloned receptor and the first evidence that NT-induced cGMP accumulation requires the product ion of nitric oxide.