DOES NITRIC-OXIDE REGULATE CAPACITATIVE CA INFLUX IN HEK-293 CELLS

It has been proposed that capacitative Ca influx into both pancreatic acinar cells and HT-29 colonic cells is regulated by stimulation of ni tric oxide synthase (NOS). NO, in turn, controls cGMP levels through e ffects on guanylate cyclase. We tested this possibility by measuring C a (and Pa) entry into human embryonic kidney 293 cells and into 293 ce lls that had been transfected with the neuronal NOS gene (293/NOS). 29 3 cells had undetectable levels of NOS, while 293/NOS cells exhibited very high levels [Bredt D.S., Ferris C.D., Snyder S.H. Nitric oxide sy nthase regulatory sites. J Biol Chem 1992; 267: 10976-10981]. Ca (or B a) entry into single cells was measured as the rate of increase of the Fura-2 fluorescence ratio (digital imaging microscopy) during rapid c hanges from Ca-free (or Ba-free) to Ca- (or Ba-) containing solutions (using high K to depolarize the membrane potential). cGMP levels (EIA method) were measured to correlate to rates of Ca entry. 100 mu M ATP caused release of Ca from internal stores, but no sustained plateau du e to Ca entry in either 293 or 293/NOS cells. Cyclopiazonic acid (CPA, which inhibits the Ca pump of the internal store, allowing Ca to leak from the store) caused apparent Ca entry to increase 5-10-fold from s imilar, low levels in both 293 and 293/NOS cells. CPA-stimulated Ca en try was unaffected by the NOS inhibitor N-nitro-L-arginine (L-NA) in e ither 293 or 293/NOS cells. In 293 cells [cGMP] was low; ATP; and CPA both increased [cGMP] by 2-fold, and the guanylate cyclase inhibitor L Y83583 and L-NA decreased [cGMP] by 50-75%. [cGMP] was 20-fold higher in 293/NOS cells than in 293 cells; these [cGMP] were not affected by ATP and CPA, but were effectively decreased by 80-90% by L-NA and by L Y83583. Thus, [cGMP] and Ca or Pa entry showed no relationship to each other: Ca entry was small into cells in which [cGMP] was either low ( resting 293, CPA + L-NA or CPA + LY83583), intermediate (ATP-treated 2 93) or high (resting 293/NOS). Similarly, Ca entry was high into cells in which [cGMP] was low (CPA + L-NA- or CPA + LY83583-treated 293), i ntermediate (CPA-treated 293 and CPA + L-NA-treated 293/NOS) or high ( CPA- or ATP-treated 293/NOS). We conclude that, as in most other non-e xcitable cells, Ca entry into 293 cells is stimulated by loss of Ca fr om the store but, unlike pancreatic and colonic cells, this capacitati ve Ca entry does not appear to be regulated by NO and cGMP Therefore, although capacitative entry across the plasma membrane may be regulate d by NO and cGMP in GI epithelial cells, this regulation does not occu r in all cells.