A Simple and Rapid Method for Standard Preparation of Gas Phase Extract of Cigarette Smoke

Cigarette smoke consists of tar and gas phase: the latter is toxicologically important because it can pass through lung alveolar epithelium to enter the circulation. Here we attempt to establish a standard method for preparation of gas phase extract of cigarette smoke (CSE). CSE was prepared by continuously sucking cigarette smoke through a Cambridge filter to remove tar, followed by bubbling it into phosphate-buffered saline (PBS). An increase in dry weight of the filter was defined as tar weight. Characteristically, concentrations of CSEs were represented as virtual tar concentrations, assuming that tar on the filter was dissolved in PBS. CSEs prepared from smaller numbers of cigarettes (original tar concentrations = 20 mg/ml), the curves were shifted rightward. Accordingly, the cytotoxic activity was detected in PBS of the second reservoir downstream of the first one with larger numbers of cigarettes. CSEs prepared from various cigarette brands showed comparable concentration-response curves for cytotoxicity. Two types of CSEs prepared by continuous and puff smoking protocols were similar regarding concentration-response curves for cytotoxicity, pharmacology of their cytotoxicity, and concentrations of cytotoxic compounds. These data show that concentrations of CSEs expressed by virtual tar concentrations can be a reference value to normalize their cytotoxicity, irrespective of numbers of combusted cigarettes, cigarette brands and smoking protocols, if original tar concentrations are <= 15 mg/ml

Agonist-promoted Ubiquitination Differentially Regulates Receptor Trafficking of Endothelin Type A and Type B Receptors

Two types of G protein-coupled receptors for endothelin-1 (ET-1), ET type A receptor (ETAR) and ETBR, closely resemble each other, but upon ET-1 stimulation, they follow totally different intracellular trafficking pathways; ETAR is recycled back to plasma membrane, whereas ETBR is targeted to lysosome for degradation. However, the mechanisms for such different fates are unknown. Here we demonstrated that ETBR but not ETAR was ubiquitinated on the cell surface following ET-1 stimulation and that ETBR was internalized and degraded in lysosome more rapidly than ETAR. The mutant ETBR (designated 5KR mutant) in which 5 lysine residues in the C-tail were substituted to arginine was not ubiquitinated, and its rates of internalization and degradation after ET-1 stimulation became slower, being comparable with those of ETAR. Confocal microscopic study showed that following ET-1 stimulation, ETAR and 5KR mutant of ETBR were co-localized mainly with Rab11, a marker of recycling endosome, whereas ETBR was co-localized with Rab7, a marker of late endosome/lysosome. In the 5KR mutant, ET-1-induced ERK phosphorylation and an increase in the intracellular Ca2+ concentration upon repetitive ET-1 stimulation were larger. A series of ETBR mutants (designated 4KR mutant), in which either one of 5 arginine residues of the 5KR mutant was reverted to lysine, were normally ubiquitinated, internalized, and degraded, with ERK phosphorylation being normalized. These results demonstrate that agonist-induced ubiquitination at either lysine residue in the C-tail of ETBR but not ETAR switches intracellular trafficking from recycling to plasma membrane to targeting to lysosome, causing decreases in the cell surface level of ETBR and intracellular signaling

Pharmacological properties of cytotoxic activities of two types of gas phase extracts of cigarette smoke.

<p>The gas phase extracts of cigarette smoke at the virtual tar concentration of 10 mg/ml PBS were prepared from Hi-Lite brand cigarettes by either continuous (cCSE) or puff smoking protocol (pCSE), and they were subjected to MTS reduction assay (A) and LDH leakage assay (B). For determination of the effects of inhibitors of protein kinase C or NADPH oxidase, 5 µM BIS I or 1 µM DPI was added to the culture medium of C6 glioma cells, respectively, 30 min before the start of 4-h incubation with cCSE or pCSE. In panel A, MTS reduction activity was represented as a percentage of the control value in the absence of CSEs (PBS) within the vehicle-treated group. In panel B, LDH activity leaked into culture medium was represented as a percentage of total activity in the medium of cells lysed by 0.2% Triton X-100. Values represent means ± SE of three experiments, each in triplicate. **P<0.01 vs PBS-treated cells within either of three groups (Vehicle-, BIS I- and DPI-treated groups); ^##P<0.01 vs cCSE- or pCSE-treated cells within the vehicle-treated group.</p

Sensitivities of various cultured cells to the gas phase extracts of cigarette smoke.

<p>The gas phase extracts of cigarette smoke (cCSE) at the virtual tar concentration of 10 mg/ml were prepared from Hi-Lite brand cigarettes by continuous smoking protocol, and they were subjected to MTS reduction assay using various cultured cells. MTS reduction activity was represented as a percentage of the control value in the absence of cCSE. Values represent means ± SE of three experiments, each in triplicate. (A) C6, rat glioma cells; HEK293T, human embryonic kidney cells; CHO, Chinese hamster ovary cells; HeLa, human cervical carcinoma cells; U937, human monocytes; RAW264.7, mouse macrophages; HUVEC, immortalized human umbilical vein endothelial cells; A7r5, rat aorta smooth muscle cells. (B) SBC-3, human lung small cell carcinoma; H1299, human lung squamous cell carcinoma; A549, human lung adenocarcinoma.</p

Quantification of the weight of the tar of cigarette smoke trapped on the Cambridge filter.

<p>(A) Time-course of a decrease in the weight of the tar phase of cigarette smoke trapped on the Cambridge filter after drying at 25°C (open circle) or 55°C (closed circle). Four cigarettes of Hi-Lite brand were sequentially combusted and the main-stream smoke was sucked through a Cambridge filter at a constant flow rate of 1.050 l/min by an aspiration pump. After combustion of cigarette, the filter was dried for various lengths of time at 25°C (open circle) or 55°C (closed circle), and the weight of the tar phase of cigarette smoke trapped on the Cambridge filter was obtained by subtracting the filter weight before combustion of cigarette from the weight after combustion. (B) The relationship between the number of combusted cigarettes and the dry weight of the tar phase trapped on the Cambridge filter. Various numbers of Hi-Lite brand cigarettes were sequentially combusted as described in A. After combustion, the filter was dried for 12 h at 25°C, and the dry weight of the tar phase on the Cambridge filter was determined as described in A. Values represent means ± SD of three experiments. *, P<0.05; **, P<0.01 versus 25°C.</p

Tar content per cigarette of various brands, the dry weight of tar trapped on the Cambridge filter after combustion of one cigarette and the EC₅₀ values of cCSEs for inhibition of MTS reduction activity.

a<p>Nicotine and tar content per cigarette is the value reported by its manufacturer and determined by puff smoking based on ISO regulation.</p>b<p>For determination of dry tar weight per cigarette, smoke of one cigarette from either brand was continuously sucked through the Cambridge filter, and the increase in the dry weight of the filter was determined (represented as means ± SD of three experiments).</p>c<p>For determination of the EC₅₀ values, cCSEs at the original tar concentration of 10 mg/ml were prepared from cigarettes of various brands by continuous smoking, and subjected to MTS reduction assay in C6 glioma cells for construction of concentration-response curves from which the EC₅₀ values (means ± SE of three experiments, each in triplicate) were determined.</p><p>Tar content per cigarette of various brands, the dry weight of tar trapped on the Cambridge filter after combustion of one cigarette and the EC₅₀ values of cCSEs for inhibition of MTS reduction activity.</p