N(omega)-nitro-L-arginine decreases resting cytosolic [Ca2+] and enhances heat stress-induced increase in cytosolic [Ca2+] in human colon carcinoma T84 cells

N(omega)-nitro-L-arginine (LNNA) inhibits the synthesis of heat shock proteins in animals and cultured cells exposed to heat stress. Heat shock protein synthesis is known to be Ca2+-dependent. In this study, we have characterized the effect of LNNA on Ca2+]i before and after heat stress in human colon carcinoma T84 cells. In untreated cells incubated in the presence of external Ca2+, the resting Ca2+]i was 201+/-3 nM. If these cells were exposed to 45 degrees C for 10 min, Ca2+]i increased by 50+/-2%. Preincubation with LNNA (100 microM) without subsequent heating led to a decrease in Ca2+]i in a LNNA concentration-dependent manner. Preincubation with LNNA followed by heating increased Ca2+]i to levels 88+/-5% greater than cells heated without LNNA pretreatment. Incubating cells in medium without external Ca2+ (no heating, no LNNA treatment) lowered resting Ca2+]i to 115+/-2 nM and greatly reduced the increase in Ca2+]i observed if cells were heated in the presence of Ca2+, indicating that external Ca2+ plays an important role in the maintenance of Ca2+]i in T84 cells. With external Ca2+ absent, LNNA pretreatment further reduced Ca2+]i in unheated cells, and heating failed to enhance Ca2+]i. We determined (with external Ca2+ present) that the heat-stress induced increase in Ca2+]i in T84 cells was blocked by dichlorobenzamil, a Na+/Ca2+ exchanger inhibitor, suggesting that the exchanger mediates Ca2+ entry. The median inhibitory concentration (IC50) in cells not treated with LNNA was 0.970+/-0.028 microM. With LNNA pretreatment, the IC50 was 5.099+/-0.107 microM. Heat stress of T84 cells did not affect the binding affinity of the Na+/Ca2+ exchanger for external Ca2+, but it increased the maximal velocity of the exchanger. In unheated cells, preincubation with LNNA decreased the binding affinity of the exchanger for Ca2+, but after heat treatment, both the binding affinity and maximal velocity of the exchanger increased. Our data are consistent with the idea that LNNA affects the activity of the Na+/Ca2+ exchanger. We also determined there are intracellular Ca2+ pools in T84 cells sensitive to thapsigargin, monensin, and ionomycin. Treatment with TMB-8, a blocker of Ca2+ sequestration and mobilization, or ionomycin inhibited the LNNA-induced decrease in Ca2+]i observed in the absence of external Ca2+, suggesting that LNNA promotes Ca2+ sequestration.