Citations

Abstract Previous studies have suggested that the proinflammatory cytokine, TNF-α, contributes to airway hyperresponsivness by altering airway smooth muscle (ASM) Ca2+ responses to agonist stimulation. The present study examined the effects of TNF-α on Ca2+ influx pathways in cultured human ASM cells (HASMCs). Proteins encoded by the transient receptor potential (TRP) gene family function as channels through which receptor-operated and store-operated Ca2+ entry (SOCE) occur. In the present study, the presence of TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 mRNA and protein expression was confirmed in cultured HASMCs using RT-PCR and Western blot analysis. TNF-α treatment significantly increased TRPC3 mRNA and protein levels in HASMCs as well as SOCE. TNF-α treatment also increased both the peak and plateau intracellular Ca2+ concentration responses in HASMCs elicited by acetylcholine and bradykinin. The effects of TNF-α treatment on SOCE and agonist-induced intracellular Ca2+ concentration responses were attenuated using small interfering RNA transfection, which knocked down TRPC3 expression. Thus, in inflammatory airway diseases, TNF-α treatment may result in increased myocyte activation due to altered Ca2+ influx pathways. These results suggest that TRPC3 may be an important therapeutic target in inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease.

Abstract Purpose: According to some studies, susceptibility of cells to anticancer drug-induced apoptosis is markedly inhibited by targeted deletion of genes encoding apoptotic protease activating factor 1 (Apaf-1) or certain caspases. Information about levels of these polypeptides in common cancer cell types and any possible correlation with drug sensitivity in the absence of gene deletion is currently fragmentary. Experimental Design: Immunoblotting was used to estimate levels of Apaf-1 as well as procaspase-2, -3, -6, -7, -8, and -9 in the 60-cell-line panel used for drug screening by the National Cancer Institute. Sensitivity of the same lines to >80,000 compounds was determined with 48-hour sulforhodamine B binding assays. Additional 6-day assays were performed for selected agents. Results: Levels of Apaf-1 and procaspases varied widely. Apaf-1 and procaspase-9, which are implicated in caspase activation after treatment of cells with various anticancer drugs, were detectable in all of the cell lines, with levels of Apaf-1 ranging from ∼1 × 105 to 2 × 106 molecules per cell and procaspase-9 from ∼5 × 103 to ∼1.6 × 105 molecules per cell. Procaspase-8 levels ranged from 1.7 × 105 to 8 × 106 molecules per cell. Procaspase-3, a major effector caspase, varied from undetectable to ∼1.6 × 106 molecules per cell. Correlations between levels of these polypeptides and sensitivity to any of a variety of experimental or conventional antineoplastic agents in either 2-day or 6-day cytotoxicity assays were weak at best. Conclusions: With the exception of caspase-3, all of the components of the core cell-death machinery are expressed in all of the cell lines examined. Despite variations in expression, levels of any one component are not a major determinant of drug sensitivity in these cells in vitro.

AbstractSummary: The trace metal copper is an essential cofactor for a number of biological processes, including mitochondrial oxidative phosphorylation, free‐radical eradication, neurotransmitter synthesis and maturation, and iron metabolism. Consequently, copper transport at the cell surface and the delivery of copper to intracellular proteins are critical events in normal cellular homeostasis. Four genes have been reported to influence the cellular uptake and the delivery of copper to specific cell compartments and proteins. These include hCTR1, which regulates cellular copper uptake; HAH1, which mediates the transfer of copper to the Menkes and Wilson disease transporters; CCS, which is related to the transfer of copper to superoxide dismutase; and hCOX17, which directs trafficking of copper to mitochondrial cytochrome‐c oxidase. At present, no genetic disorders have been associated with defects in these four copper transporter genes. In this study, we test the possibility that defective copper uptake or intracellular translocation represents the basic defect in three categories of candidate phenotypes among 22 patients: ethylmalonic encephalopathy; mitochondriopathies of unknown aetiology; and neurodevelopmental abnormalities with clinical and chemical evidence of copper deficiency. Mutation analyses of the copper uptake protein, hCTR1, and the three copper chaperones were performed by direct sequencing of the whole coding regions. No causative mutations were identified for the four copper transporter genes in 22 patients. A heterozygous polymorphism (847G>A) for CCS was detected in 7 patients. For the distinct disease entity ethylmalonic encephalopathy, we additionally show normal mRNA levels for each of the four genes. The negative results notwithstanding, we encourage ongoing study of additional patients with candidate phenotypes. Further, our results are consistent with the notion that other unknown copper‐related transporters could be involved in diseases.

Carbon monoxide (CO) is proposed as a physiological messenger. CO activates cGMP and has a direct effect on potassium channels. Both actions of CO lead to hyperpolarization of a cell's resting membrane potential, suggesting that CO may function as a hyperpolarizing factor, although direct evidence is still lacking. Here we take advantage of the known membrane potential gradient that exists in the muscle layers of the gastrointestinal tract to determine whether CO is an endogenous hyperpolarizing factor. We find that heme oxygenase-2-null mice have depolarized smooth muscle cells and that the membrane potential gradient in the gut is abolished. Exogenous CO hyperpolarizes the membrane potential. Regions of the canine gastrointestinal tract that are more hyperpolarized generate more CO and have higher heme oxygenase activity than more depolarized regions. Our results suggest that CO is a critical hyperpolarizing factor required for the maintenance of intestinal smooth muscle membrane potential and gradient.

Nitric oxide (NO) and carbon monoxide (CO) seem to be neurotransmitters in the brain. The colocalization of their respective biosynthetic enzymes, neuronal NO synthase (nNOS) and heme oxygenase-2 (HO2), in enteric neurons and altered intestinal function in mice with genomic deletion of the enzymes ( nNOS Δ/Δ and HO2 Δ/Δ ) suggest neurotransmitter roles for NO and CO in the enteric nervous system. We now establish that NO and CO are both neurotransmitters that interact as cotransmitters. Small intestinal smooth muscle cells from nNOS Δ/Δ and HO2 Δ/Δ mice are depolarized, with apparent additive effects in the double knockouts ( HO2 Δ/Δ / nNOS Δ/Δ ). Muscle relaxation and inhibitory neurotransmission are reduced in the mutant mice. In HO2 Δ/Δ preparations, responses to electrical field stimulation are nearly abolished despite persistent nNOS expression, whereas exogenous CO restores normal responses, indicating that the NO system does not function in the absence of CO generation.