Strain-related differences in the pneumotoxic effects of chronically administered butylated hydroxytoluene on protein kinase C and calpain.

BALB/cBy (BALB) and CXB H mice responded similarly to a single intraperitoneal injection of butylated hydroxytoluene (BHT). This transient pneumotoxicity was characterized by an elevated lung weight and alveolar damage. These changes were accompanied by alterations in the calcium second messenger pathway, namely, two- to five-fold decreases in the activities and pulmonary content of protein kinase C alpha (PKC alpha) and calcium-dependent protease isozyme II (calpain II). BALB and CXB H mice varied in their responsiveness to a chronic (4-6 weekly injections) BHT regimen. CXB H mice became tolerant to BHT and all of the above parameters had returned to control values when examined after the last injection. Chronic BHT administration also failed to enhance lung tumor multiplicity in CXB H mice when the first BHT injection was preceded by the carcinogen, urethane. In contrast, the additional BHT doses potentiated the pathological and biochemical alterations in BALB mice above that found with acute treatment. This included a chronic inflammatory response characterized by the presence of activated macrophages, and a lung tumor multiplicity that was 3-fold greater than in BALB mice receiving urethane alone. One BHT injection increased calpain II mRNA in both strains (1.5- to 3-fold); mRNA declined following multiple BHT injections in BALB mice, but remained elevated in CXB H mice. Studies with the cytochrome P450 inhibitor, piperonyl butoxide, indicated that metabolism of BHT was required for both its acute and chronic effects. We conclude the following: (i) Differences between inbred mice in their susceptibility to chronic pneumotoxicant exposure may exist even when they respond similarly to an acute exposure of the same agent; (ii) A chronic inflammatory state involving a high concentration of activated macrophages may play an important role in tumor enhancement by a non-carcinogen; (iii) The protein contents of PKC alpha and calpain II decrease during BHT-induced lung damage.

Calpain-induced downregulation of activated protein kinase C-alpha affects lung epithelial cell morphology.

A few minutes after mouse lung epithelial cell lines were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), the cells rounded up and pulled away from their neighbors. Several hours later, the cells flattened out to resume their original morphology. To begin to characterize the enzymology underlying these changes, the subcellular distribution and intracellular content of the TPA receptor, protein kinase C (PKC), and its putative endogenous regulator, the Ca(2+)-dependent protease, calpain, were investigated. Of eight PKC isozymes examined in several tumorigenic and nontumorigenic cell lines, all cells contained PKC-alpha, PKC-delta, and PKC-zeta. TPA rapidly (5 min) translocated PKC-alpha from the cytosol to the particulate fraction; PKC-alpha concentrations then decreased with continued TPA exposure. PKC-zeta levels and intracellular location were not affected. An inhibitor of PKC activity, GF 109203X, prevented the initial morphological change. The calpain II isozyme was also found in all cell lines, and its cellular content increased as a result of TPA treatment. Calpain inhibitor I did not affect the initial shape change but prevented subsequent flattening of the cells. We therefore conclude that PKC activation is required for the TPA-induced alterations in lung cell morphology and that calpain mediates the return to a flattened epithelial appearance.

The effects of calcium site occupancy and reagent length on reactivity of calmodulin lysyl residues with heterobifunctional aryl azides. Mapping interaction domains with specific calmodulin photoprobe derivatives.

The relationship of structural and functional moieties on calmodulin is important in all venues of cell activity. In this study, we investigate the effect of lysine modification on calmodulin function. Azidosalicylate reagents containing different "linker arm" lengths, between the photoactive terminus and an amine-reactive N-hydroxysuccinimidyl ester moiety were used to modify calmodulin lysines at three different positions in a calcium-dependent manner. The short cross-linker, (ASNE-2 (where ASNE represents azidosalicylate N-hydroxysuccinimidyl ester), modifies Lys-75, whereas the longer reagent, ASNE-6, modifies lysines 21, 75, and 94. The modification of these different lysines is shown to be calcium-dependent. At 1-100 microM levels of calcium, only Lys-94 is modified, suggesting that modification of this residue is directed by both the binding of calcium to calcium-binding loops III and IV and the hydrophobic pocket exposed between these two loops as a result of calcium binding. At higher calcium concentrations (> 200 microM), where sites I and II become filled, modification of Lys-21 or Lys-75 also was observed. All the modified calmodulins were able to stimulate 3',5'-cyclic-nucleotide phosphodiesterase fully although the Kact for the Lys-75 and Lys-21 derivatives increased 10- and 50-fold, respectively. None of the modifications affected the activation of erythrocyte plasma membrane Ca(2+)-ATPase. Only the ASNE-6 Lys-75 derivative showed efficient (40%) photocross-linking to the Ca(2+)-ATPase. The ASNE-2 Lys-75 derivative as well as the ASNE-6 Lys-21 and Lys-94 derivatives did not show efficient calcium-dependent photocross-linking to this enzyme.

Heterobifunctional cross-linking agents incorporating perfluorinated aryl azides.

New heterobifunctional cross-linking reagents were developed that possess a photoactive tetrafluorinated phenyl azide as the photoactive terminus and a chemically reactive succinimidyl ester as the electrophilic terminus. These reagents, succinimidyl N-(4-azido-2,3,5,6-tetrafluorobenzoyl)tyrosinate (9) and succinimidyl 2-(4-azido-2,3,5,6-tetrafluorophenyl)thiazole-4-carboxylate (15), were designed to possess either an 125I or 35S radiolabel, respectively. In a biochemical study, the latter reagent was coupled to Lys-75 of calmodulin (CaM), and the radioiodinated monoadduct was photochemically cross-linked, in a calcium-dependent manner, to the porcine erythrocyte plasma membrane Ca2+, Mg2(+)-ATPase. Densitometry scans of the gel indicated a reproducible 22% cross-linking of the CaM with one of the Ca2+,Mg2(+)-ATPase bands. Since the purification of the Ca2+,Mg2(+)-ATPase results in micelles having Ca2+,Mg2(+)-ATPase with its CaM binding site oriented both to the inside and outside of the micelle, the amount of Ca2+,Mg2(+)-ATPase available for cross-linking was reduced by approximately half, suggesting that the actual cross-linking efficiency was on the order of 40%.

Photoaffinity heterobifunctional cross-linking reagents based on N-(azidobenzoyl)tyrosines.

New heterobifunctional cross-linking reagents that possessed a photoactive terminus, an electrophilic terminus, and a linking arm between the two termini that had a radiolabeled, enzymatically cleavable bond were synthesized. In a model study, succinimidyl N-[N'-(4-azidobenzoyl)tyrosyl]-beta-alanate (16A) was coupled to n-butylamine (a Lys surrogate), iodinated, and cleaved with chymotrypsin in the presence of tyrosylamide to afford the desired adduct (N-(N'-(4-azidobenzoyl)-3-iodotyrosyl)tyrosinamide, thereby demonstrating the feasibility of the enzymatic cleavage. In a biochemical study, succinimidyl N-[N'-(3-azido-5-nitrobenzoyl)tyrosyl]-beta-alanate (16C) was coupled to Lys-75 of calmodulin (CaM), and the radioiodinated monoadduct was successfully photo-cross-linked, in a calcium-dependent manner, to the human erythrocyte plasma membrane Ca2+,Mg2(+)-ATPase and to a synthetic fragment (M13) containing the CaM-binding region of myosin light-chain kinase. In the latter case, densitometry readings indicated 20% cross-linking efficiency.

Photoaffinity heterobifunctional cross-linking reagents based on azide-substituted salicylates.

Photoaffinity heterobifunctional cross-linking reagents are described that incorporate a 4-azidosalicylate group at one terminus and an N-hydroxysuccinimidyl ester at the other terminus with a "linking arm" of variable length separating the photoactive and electrophilic termini. Exposure of calmodulin (CaM) to succinimidyl N-[2-[(4-azidosalicyloyl)oxy]ethyl]suberamate (4C) led to monoadducts at Lys-21, Lys-75, and Lys-94. Separation of the monoadducts from CaM and polyadducts, radioiodination, and photolysis in the presence of human erythrocyte plasma membrane Ca2+, Mg2(+)-ATPase led to calcium-dependent cross-linking with 8% cross-linking efficiency.