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

A possible role of the nitric oxide (NO)/cGMP pathway in the regulation of Ca2+ entry into HT291B6 human colonic epithelial cells was investigated using digital image processing of Fura-2 fluorescence and immunoblotting for nitric oxide synthase (NOS). We tested the hypothesis that 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 depolarize the cell membrane potential and thereby exclude effects of NO and CGMP on K+ or Cl- channels, which might affect Ca2+ entry. Sodium nitroprusside (SNP, 0.5 mu M and 30 mu M), a NO donor, only slightly raised intracellular [Ca2+] ([Ca2+](i)) in resting cells, but in 100 mu M carbachol-stimulated cells the sustained, elevated Ca2+ plateau (reflecting Ca2+ entry) as well as Ba2+ entry were increased by 0.5 mu M SNP, 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 capacitative Ca2+ entry was activated by carbachol or cyclopiazonic acid (10 mu M). The GC inhibitor LY83583 (10 mu M) reduced carbachol-stimulated Ca2+ entry, and this entry was restored with 8-Br-cGMP. Western blotting revealed that endothelial-type NOS was present in the particulate fraction of cells. The data are consistent with the notion that Ca2+ 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 from the store during the emptying process, but NO and cGMP have only small effects of their own to open the Ca2+ channel in the plasma membrane. High [SNP] appeared to be inhibitory while low [SNP] was stimulatory, indicating that a precise range of [NO] may be required for effective stimulation of Ca2+ entry.