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.
Kiang
J G
JG
Department of Cellular Injury, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA. Dr._Juliann_Kiang@WRSMTP-ccmail.army.mil
McClain
D E
DE
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000493:pharmacokinetics*; D002451:Cell Compartmentation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D018869:Heat-Shock Response / Q000187:drug effects*; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D008985:Monensin / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism; D019335:Nitroarginine / Q000494:pharmacology*; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000201:enzymology
D004791:Enzyme Inhibitors; D007476:Ionophores; D019831:Sodium-Calcium Exchanger; C041470:3',4'-dichlorobenzamil; D019335:Nitroarginine; D019284:Thapsigargin; D000584:Amiloride; D008985:Monensin; D019001:Nitric Oxide Synthase; D002118:Calcium