POSSIBLE REGULATION OF CAPACITATIVE CA2-CELLS BY NO AND CGMP( ENTRY INTO COLONIC EPITHELIAL)

A possible role of the nitric oxide (NO)/cGMP pathway in the regulatio n of Ca2+ entry into HT291B6 human colonic epithelial cells was invest igated using digital image processing of Fura-2 fluorescence and immun oblotting for nitric oxide synthase (NOS). We tested the hypothesis th at Ca2+ store depletion causes increased NOS activity and [NO], which is stimulatory to Ca2+ entry by increasing guanylate cyclase (GC) and [cGMP]. Cells were incubated in 95 mM K+-containing solutions to depol arize the cell membrane potential and thereby exclude effects of NO an d CGMP on K+ or Cl- channels, which might affect Ca2+ entry. Sodium ni troprusside (SNP, 0.5 mu M and 30 mu M), a NO donor, only slightly rai sed intracellular [Ca2+] ([Ca2+](i)) in resting cells, but in 100 mu M carbachol-stimulated cells the sustained, elevated Ca2+ plateau (refl ecting Ca2+ entry) as well as Ba2+ entry were increased by 0.5 mu M SN P, while 5, 10 or 30 mu M SNP either had no effect or were inhibitory. Pretreatment of cells with the NOS inhibitor N-nitro-L-arginine (1 mM ) reduced carbachol-stimulated Ca2+ entry, and simultaneous treatment with 0.5 mu M (but not 30 mu M) SNP restored Ca2+ influx. 8-Br-cGMP (1 mM) had little effect on [Ca2+](i) or on rates of Ca2+ or Ba2+ influx into resting cells, but there were large effects on cells in which ca pacitative Ca2+ entry was activated by carbachol or cyclopiazonic acid (10 mu M). The GC inhibitor LY83583 (10 mu M) reduced carbachol-stimu lated Ca2+ entry, and this entry was restored with 8-Br-cGMP. Western blotting revealed that endothelial-type NOS was present in the particu late fraction of cells. The data are consistent with the notion that C a2+ entry into HT29/B6 cells is regulated by endothelial NOS/NO and GC /cGMP, but effects are most pronounced in store-depleted cells. Thus, NO and cGMP appear to potentiate the action of messengers released fro m the store during the emptying process, but NO and cGMP have only sma ll effects of their own to open the Ca2+ channel in the plasma membran e. High [SNP] appeared to be inhibitory while low [SNP] was stimulator y, indicating that a precise range of [NO] may be required for effecti ve stimulation of Ca2+ entry.