Drp1 is dispensable for apoptotic cytochrome c release in primed MCF10A and fibroblast cells but affects Bcl-2 antagonist-induced respiratory changes.

BACKGROUND AND PURPOSE: Dynamin-related protein 1 (Drp1) mediates mitochondrial fission and is thought to promote Bax/Bak-induced cytochrome c release during apoptosis. Conformationally active Bax, Bak and Bax/Bak-activating BH3-only proteins, such as Bim, are restrained by anti-apoptotic Bcl-2 proteins in cells that are 'primed for death'. Inhibition of Bcl-2/Bcl-xL/Bcl-w by the antagonist ABT-737 causes rapid apoptosis of primed cells. Hence, we determined whether Drp1 is required for cytochrome c release, respiratory alterations and apoptosis of cells that are already primed for death. EXPERIMENTAL APPROACH: We tested the Drp1 inhibitor mdivi-1 for inhibition of cytochrome c release in MCF10A cells primed by Bcl-2 overexpression. We measured ATP synthesis-dependent, -independent and cytochrome c-limited maximal oxygen consumption rates (OCRs) and cell death of immortalized wild-type (WT) and Drp1 knockout (KO) mouse embryonic fibroblasts (MEFs) treated with ABT-737. KEY RESULTS: Mdivi-1 failed to attenuate ABT-737-induced cytochrome c release. ABT-737 decreased maximal OCR measured in the presence of uncoupler in both WT and Drp1 KO MEF, consistent with respiratory impairment due to release of cytochrome c. However, Drp1 KO MEF were slightly less sensitive to this ABT-737-induced respiratory inhibition compared with WT, and were resistant to an initial ABT-737-induced increase in ATP synthesis-independent O2 consumption. Nevertheless, caspase-dependent cell death was not reduced. Pro-apoptotic Bax was unaltered, whereas Bak was up-regulated in Drp1 KO MEF. CONCLUSIONS AND IMPLICATIONS: The findings indicate that once fibroblast cells are primed for death, Drp1 is not required for apoptosis. However, Drp1 may contribute to ABT-737-induced respiratory changes and the kinetics of cytochrome c release.

Bcl-2 family interaction with the mitochondrial morphogenesis machinery.

The regulation of both mitochondrial dynamics and apoptosis is key for maintaining the health of a cell. Bcl-2 family proteins, central in apoptosis regulation, also have roles in the maintenance of the mitochondrial network. Here we report that Bax and Bak participate in the regulation of mitochondrial fusion in mouse embryonic fibroblasts, primary mouse neurons and human colon carcinoma cells. To assess how Bcl-2 family members may regulate mitochondrial morphogenesis, we determined the binding of a series of chimeras between Bcl-xL and Bax to the mitofusins, mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2). One chimera (containing helix 5 (H5) of Bax replacing H5 of Bcl-xL (Bcl-xL/Bax H5)) co-immunoprecipitated with Mfn1 and Mfn2 significantly better than either wild-type Bax or Bcl-xL. Expression of Bcl-xL/Bax H5 in cells reduced the mobility of Mfn1 and Mfn2 and colocalized with ectopic Mfn1 and Mfn2, as well as endogenous Mfn2 to a greater extent than wild-type Bax. Ultimately, Bcl-xL/Bax H5 induced substantial mitochondrial fragmentation in healthy cells. Therefore, we propose that Bcl-xL/Bax H5 disturbs mitochondrial morphology by binding and inhibiting Mfn1 and Mfn2 activity, supporting the hypothesis that Bcl-2 family members have the capacity to regulate mitochondrial morphology through binding to the mitofusins in healthy cells.

Polyflex self-expanding, removable plastic stents: assessment of treatment efficacy and safety in a variety of benign and malignant conditions of the esophagus.

BACKGROUND: Historically, esophageal fistulas, perforations, and benign and malignant strictures have been managed surgically or with the placement of permanent endoprostheses or metallic stents. Recently, a removable, self-expanding, plastic stent has become available. The authors investigated the use of this new stent at their institution. METHODS: The study reviewed all the patients who received a Polyflex stent for an esophageal indication at the authors' institution between January 2004 and October 2006. Duration of placement, complications, and treatment efficacy were recorded. RESULTS: A total of 37 stents were placed in 30 patients (14 women and 16 men) with a mean age of 68 years (range, 28-92 years). Stent placement included 7 for fistulas, 3 for perforations, 1 for an anastomotic leak, 7 for malignant strictures, and 19 for benign strictures (8 anastomotic, 1 caustic, 5 reflux, 2 radiation, and 2 autoimmune esophagitis strictures, and 1 post-Nissen gas bloat stricture). The mean follow-up period was 6 months. Stent deployment was successful for all the patients, and no complications resulted from stent placement or removal. Nine stents migrated spontaneously. Three of three perforations and three of five fistulas sealed. Only one stent was removed because of patient discomfort. One patient with a radiation stricture experienced tracheoesophageal fistulas secondary to pressure necrosis. Of 20 patients with stricture, 18 experienced improvement in their dysphagia. CONCLUSION: Self-expanding, removable plastic stents are easily and safely placed and removed from the esophagus. This has facilitated their use in the authors' institution for an increasing number of esophageal conditions. Further studies to help define their ultimate role in benign and malignant esophageal pathology are warranted.

Dynamics of mitochondrial morphology in healthy cells and during apoptosis.

Mitochondria exist as dynamic networks that often change shape and subcellular distribution. The number and morphology of mitochondria within a cell are controlled by precisely regulated rates of organelle fusion and fission. Recent reports have described dramatic alterations in mitochondrial morphology during the early stages of apoptotic cell death, a fragmentation of the network and the remodeling of the cristae. Surprisingly, proteins discovered to control mitochondrial morphology appear to also participate in apoptosis and proteins associated with the regulation of apoptosis have been shown to affect mitochondrial ultrastructure. In this review the recent progress in understanding the mechanisms governing mitochondrial morphology and the latest advances connecting the regulation of mitochondrial morphology with programmed cell death are discussed.

Structural changes of mitochondria related to apoptosis.

The original concept of apoptosis stressed the morphological changes of the nucleus, condensation with the aggregation of chromatin, and the intactness of intracellular organelles including mitochondria. However, the application of molecular biology and flow-cytometric techniques to the research field of apoptosis has led to the proposal of the apoptotic processes which emphasizes the 'swelling of mitochondria' due to the opening of the 'permeability transition pores' of the mitochondrial membranes followed by a series of events including the collapse of the transmembrane potential of mitochondria and release of cytochrome c from mitochondria into the cytosol. Enlargement of mitochondria induced by various pathological conditions are classified into two categories: the swelling and the formation of megamitochondria (MG). Recently, we have found that free radical-induced formation of MG is succeeded by apoptotic changes of the cell. If the MG formation is actually related to apoptosis, this will be a new aspect of the structural changes of mitochondria involved in apoptosis besides the simple swelling of mitochondria. First, we will discuss the 'swelling of mitochondria' which characterizes the currently accepted hypothesis on the apoptotic processes of the cell, as described above, in the light of the literature. Second, the mechanisms controlling the size and distribution of mitochondria in the cell are dealt with paying special attention to the genetic regulation and cytoskeletons. Third, we have tried to characterize the MG formation to correlate apoptotic changes of the cell. Finally, we will discuss several problems to be solved in the future which involve mitochondria in apoptotic processes of the cell.

Opposite effects of microtubule-stabilizing and microtubule-destabilizing drugs on biogenesis of mitochondria in mammalian cells.

Distribution of mitochondria as well as other intracellular organelles in mammalian cells is regulated by interphase microtubules. Here, we demonstrate a role of microtubules in the mitochondrial biogenesis using various microtubule-active drugs and human osteosarcoma cell line 143B cells and rat liver-derived RL-34 cells. Depolymerization of microtubules by nocodazole or colchicine, as well as 2-methoxyestradiol, a natural estrogen metabolite, arrested asynchronously cultured cells in G(2)/M phase of cell cycle and at the same time inhibited the mitochondrial mass increase and mtDNA replication. These drugs also inhibited the mitochondrial mass increase in the cells that were synchronized in cell cycle, which should occur during G(1) to G(2) phase progression in normal conditions. However, stabilization of microtubules by taxol did not affect the proliferation of mitochondria during the cell cycle, yet a prolonged incubation of cells with taxol induced an abnormal accumulation of mitochondria in cells arrested in G(2)/M phase of cell cycle. Taxol-induced accumulation of mitochondria was not only demonstrated by mitochondria-specific fluorescent dyes but also evidenced by the examination of cells transfected with yellow fluorescent protein fused with mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase (pEYFP) and by enhanced mtDNA replication. Two subpopulations of mitochondria were detected in taxol-treated cells: mitochondria with high Delta(psi)(m), detectable either by Mito Tracker Red CMXRos or by Green FM, and those with low Delta(psi)(m), detectable only by Green FM. However, taxol-induced increases in the mitochondrial mass and in the level of acetylated (alpha)-tubulin were abrogated by a co-treatment with taxol and nocodazole or taxol and colchicine. These data strongly suggest that interphase microtubules may be essential for the regulation of mitochondrial biogenesis in mammalian cells.

Investigation of the substrate spectrum of the human mismatch-specific DNA N-glycosylase MED1 (MBD4): fundamental role of the catalytic domain.

The human DNA repair protein MED1 (also known as MBD4) was isolated as an interactor of the mismatch repair protein MLH1 in a yeast two-hybrid screening. MED1 has a tripartite structure with an N-terminal 5-methylcytosine binding domain (MBD), a central region, and a C-terminal catalytic domain with homology to bacterial DNA damage-specific glycosylases/lyases. Indeed, MED1 acts as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil paired with guanine. The glycosylase activity of MED1 preferentially targets G:T mismatches in the context of CpG sites; this indicates that MED1 is involved in the repair of deaminated 5-methylcytosine. Interestingly, frameshift mutations of the MED1 gene have been reported in human colorectal, endometrial, and pancreatic cancers. For its putative role in maintaining genomic fidelity at CpG sites, it is important to characterize the biochemical properties and the substrate spectrum of MED1. Here we show that MED1 works under a wide range of temperature and pH, and has a limited optimum range of ionic strength. MED1 has a weak glycosylase activity on the mutagenic adduct 3,N(4)-ethenocytosine, a metabolite of vinyl chloride and ethyl carbamate. The differences in glycosylase activity on G:U and G:T substrates are not related to differences in substrate binding and likely result from intrinsic differences in the chemical step. Finally, the isolated catalytic domain of MED1 retains the preference for G:T and G:U substrates in the context of methylated or unmethylated CpG sites. This suggests that the catalytic domain is fundamental, and the 5-methylcytosine binding domain dispensable, in determining the substrate spectrum of MED1.

Swelling of free-radical-induced megamitochondria causes apoptosis.

Recently, we have found that cultured cells from various sources exposed to free radicals become apoptotic in the presence of megamitochondria (MG). The purpose of the present study is to answer the following two questions: (1) Do functions obtained from the "MG fraction" isolated from normal mitochondria by a routine procedure represent the functions of MG since the fraction consists of enlarged and normal-size mitochondria? (2) What is the correlation between MG formation and apoptotic changes of the cell? In the present study the heavy fraction rich in mitochondria enlarged to varying degrees and the light fraction consisting mainly of normal-size mitochondria were isolated independently from the livers of rats treated with hydrazine for 4 days (4H animals) and 8 days (8H animals), and some functions related to apoptosis were compared. Results were as follows: (1) Mitochondria in both fractions obtained from 8H animals swelled far less in various media than those obtained from the controls, suggesting that the permeability transition pores had been opened before they were exposed to swelling media. (2) The membrane potential of mitochondria in both fractions obtained from 8H animals was distinctly decreased. (3) The rates of reactive oxygen species generation from mitochondria of both fractions in 4H animals were equally elevated, while those in 8H animals were equally decreased compared to those of controls. These results, together with morphological data obtained in the present study, suggest that enlarged and normal-size mitochondria are a part of MG and that the secondary swelling of MG causes the apoptotic changes in the cell.

Functional aspects of megamitochondria isolated from hydrazine- and ethanol-treated rat livers.

It is essential to analyze functions of megamitochondria (MG) to elucidate the mechanism of the formation of MG induced under various pathological conditions. The MG fraction obtained by a routine isolation procedure for normal mitochondria always consists of a mixed population of mitochondria enlarged to various degrees and also normal-sized ones. The purpose of the present study is to answer the question of whether or not data obtained from the MG fraction consisting of such a heterogeneous population of mitochondria with respect to their sizes really reflect functions of MG. In the present study mitochondria were obtained from the livers of rats treated with a 1% hydrazine diet for 8 days and those given 32% ethanol in drinking water for up to 2 months using various isolation procedures. Results obtained are summarized as follows: (i) mitochondria enlarged to various degrees and normal-sized ones are sometimes connected with each other by a narrow stalk in the hepatocyte of hydrazine-treated animals, and such connections are maintained to some extent when mitochondria are isolated; and (ii) mitochondria obtained from experimental animals by a routine isolation procedure for mitochondria ((700-7000)gR2"') and those obtained by alternative isolation procedure yielding the heavy ((500-2000)gR2"') and light ((2000-7000)gR2"') fractions show some functional similarities: decreases in the content of cytochrome a + a3; decreases in oxygen consumptions and phosphorylating abilities; decreases in monoamine oxidase and cytochrome c oxidase activities; lowered membrane potential of mitochondria; decreases in the rate of the generation of reactive oxygen species. These results may suggest that mitochondria enlarged to various degrees and normal-sized ones are functionally similar to each other and that the MG fraction obtained by a routine isolation procedure for normal mitochondria can be applied to the study of the function of MG.

Two types of the enlargement of mitochondria related to apoptosis: simple swelling and the formation of megamitochondria.

Our recent finding that free radical-induced formation of megamitochondria (MG) is followed by apoptosis has prompted us to investigate the correlation between the MG formation and the swelling of mitochondria which is considered to play a key role in early stages of apoptotic processes of the cell. Mitochondria of rat hepatocytes or RL-34 cells and those isolated from rat livers became enlarged up to three times in their diameters when they were exposed to a hypotonic medium. MG induced in the liver of rats placed on a 1% hydrazine-diet for 4-5 days or those induced in the liver of mice placed on a 2% chloramphenicol (CP)-diet for 9-10 days were endowed with a dense matrix whereas those fed with the toxic diets for longer periods of time became enlarged further and their matrix became extremely pale indicating that MG in the latter animals became swollen secondarily. The membrane potential, the content of cytochrome c and the rate of the generation of reactive oxygen species (ROS) of MG in the former animals were almost unchanged compared to those of mitochondria in control animals whereas those of MG in the latter animals became distinctly decreased. These results may suggest that free radical-induced MG possibly cause apoptosis via their secondary swelling.

Effects of coenzyme Q10 on changes in the membrane potential and rate of generation of reactive oxygen species in hydrazine- and chloramphenicol-treated rat liver mitochondria.

Effects of CoQ10 and cycloheximide (CHX) on hydrazine- and chloramphenicol (CP)-induced morphological and some functional changes of mitochondria using cultured rat hepatocytes and effects on the process of recovery from CP intoxication using mouse liver were examined. Results obtained are summarized as follows: (1) The formation of megamitochondria induced in the hepatocytes cultured for 22 h in the presence of 2 mM hydrazine or CP (300 microgram/ml) was suppressed by pretreatment of hepatocytes with CoQ10 (1 microM) or CHX (0.5 microgram/ml). This was proved by electron microscopic analysis of mitochondria. (2) Treatment of hepatocytes with hydrazine for 48 h or longer caused decreases in the membrane potential of mitochondria, which were suppressed by CoQ10. (3) Treatment of hepatocytes with hydrazine for 22 h or longer caused remarkable increases in intracellular levels of reactive oxygen species in hepatocytes, which were suppressed by CoQ10. (4) The process of recovery from the CP-induced changes of mitochondria in mouse liver was accelerated by CoQ10 and CHX.

Cycloheximide and 4-OH-TEMPO suppress chloramphenicol-induced apoptosis in RL-34 cells via the suppression of the formation of megamitochondria.

Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6, 6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity: (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH2MRos; (2) megamitochondria formation detected by staining of mitochondria with MitoTracker CMXRos under a laser confocal microscopy and electron microscopy; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2, 6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.

Free radical-induced megamitochondria formation and apoptosis.

Pathophysiological meaning and the mechanism of the formation of megamitochondria (MG) induced under physiological and pathological conditions remain obscure. We now provide evidence suggesting that the MG formation may be a prerequisite for free radical-mediated apoptosis. MG were detected in primary cultured rat hepatocytes, rat liver cell lines RL-34 and IAR-20 and kidney cell line Cos-1 treated for 22 h with various chemicals known to generate free radicals: hydrazine, chloramphenicol, methyl-glyoxal-bis-guanylhydrazone, indomethacin, H2O2, and erythromycin using a fluorescent dye Mito Tracker Red CMXRos (CMXRos) for confocal laser microscopy and also by electron microscopy. Remarkable elevations of the intracellular level of reactive oxygen species (ROS), monitored by staining of cells with a fluorescent dye carboxy-H2-DCFDA, were detected before MG were formed. Prolongation of the incubation time with various chemicals, specified above, for 36 h or longer has induced distinct structural changes of the cell, which characterize apoptosis: condensation of nuclei, the formation of apoptotic bodies, and the ladder formation. Cells treated with the chemicals for 22 h were arrested in G1 phase, and apoptotic sub-G1 populations then became gradually increased. The membrane potential of MG induced by chloramphenicol detected by CMXRos for flow cytometry was found to be decreased compared to that of mitochondria in control cells. Rates of the generation of H2O2 and O2- from MG isolated from the liver of rats treated with chloramphenicol or hydrazine were found to be lower than those of mitochondria of the liver of control animals. We suggest, based on the present results together with our previous findings, that the formation of MG may be an adaptive process at a subcellular level to unfavorable environments: when cells are exposed to excess amounts of free radicals mitochondria become enlarged decreasing the rate of oxygen consumption. Decreases in the oxygen consumption of MG may result in decreases in the rate of ROS production as shown in the present study. This will at the same time result in decreases in ATP production from MG. If cells are exposed to a large amount of free radicals beyond a certain period of time, lowered intracellular levels of ATP may result in apoptotic changes of the cell.

Complete suppresion of ethanol-induced formation of megamitochondria by 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-OH-TEMPO).

An attempt has been made to suppress the ethanol-induced formation of megamitochondria (MG) in the rat liver by 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-OH-TEMPO), a free radical scavenger, and by allopurinol (AP), a xanthine oxidase inhibitor. Changes observed in the liver of animals given ethanol (EtOH) for 1 month were remarkable decreases both in the body weight gains during the course of the experiment and in the liver weight at the time of sacrifice compared to those of the control; remarkable increases in the level of thiobarbituric acid reactive substances and lipid soluble fluorophores both in microsomes and mitochondria; decreases in the content of cytochrome a+a3 and b and lowered phosphorylating ability of mitochondria; and formation of MG in the liver. A combined treatment of animals with EtOH plus 4-OH-TEMPO completely suppressed the formation of MG in the liver induced by EtOH and distinctly improved the changes caused by EtOH, as specified above, while AP partly suppressed the MG formation. Results described herein provide additional insight into chronic hepatotoxicity of EtOH besides that previously reported. A novelty of the present work is that we were able for the first time to demonstrate reversibility of EtOH-mediated ultrastructural changes of the liver by a simple administration of aminoxyl-type free radical scavenger, 4-OH-TEMPO. Our results suggest that free radicals may be involved in the mechanism of the formation of MG induced by EtOH.

Role of free radicals in the mechanism of the hydrazine-induced formation of megamitochondria.

The effect of 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl(4-OH-TEMPO), a scavenger for free radicals, and 4-hydroxypyrazolo [3,4-d(pyrimidine)allopurinol], a xanthine oxidase inhibitor, on the hydrazine-induced changes of mitochondrial ultrastructure and those in the antioxidant system of the liver were investigated using rats as experimental animals. Animals were placed on a powdered diet containing 0.5% hydrazine for 7 d in the presence and absence of a combined treatment with 4-OH-TEMPO or allopurinol. Results obtained were as follows. 4-OH-TEMPO completely prevented the hydrazine-induced formation of megamitochondria in the liver, while it was partly prevented by allopurinol. The following changes observed in hydrazine-treated animals were improved almost completely by 4-OH-TEMPO:decreases in the body weight and liver weight; lowered rates of ADP-stimulated respiration and coupling efficiency of hepatic mitochondria; remarkable elevation of the level of lipid peroxidation. Improving effects of allopurinol were incomplete. The present results suggest that free radicals may play a key role in the mechanism of the hydrazine-induced formation of megamitochondria and that a part of free radicals generated during the hydrazine intoxication is ascribed to the degradation of purine nucleotides via xanthine oxidase. A general mechanism of the megamitochondria formation induced in various pathological conditions besides the case of hydrazine are discussed.

Induction of megamitochondria by some chemicals inducing oxidative stress in primary cultured rat hepatocytes.

Effects of hydrazine, hydrogen peroxide and bromobenzene, inducers of free radicals, and those of erythromycin and cycloheximide, inhibitors of protein synthesis on structural changes of mitochondria in primary monolayer culture of rat hepatocytes were examined using laser confocal microscope and electron microscope. After 22 h of incubation of hepatocytes with 0.2 mM hydrogen peroxide or 10 microg ml-1 of erythromycin, mitochondria became extremely enlarged. Mitochondria of hepatocytes isolated from control rats became slightly to moderately enlarged in the presence of 2 mM hydrazine, while those of hepatocytes isolated from phenobarbital-pretreated animals became extremely enlarged in the presence of 2 mM hydrazine. Cycloheximide (0.5-10.0 microg ml-1) and bromobenzene (0.1-1.0 mM) failed to induce structural changes of mitochondria. The level of cytochrome P-450 in freshly prepared hepatocytes from phenobarbital-treated rats was 2.5 times higher than that from the control rats, and remained about three times higher than the latter after 22 h of incubation with 2 mM hydrazine. The level of malondialdehyde was invariably elevated when megamitochondria were induced. These results may suggest that oxidative stress is intimately related to the mechanism of the formation of megamitochondria and that the inhibition of cytoplasmic protein synthesis seems not to contribute the phenomenon. However, the detailed mechanism by which free radicals may induce megamitochondria remains to be elucidated.

Central cholinergic effects of tricyclic antidepressants in mouse.

The apparent rate of acetylcholine (ACh) turnover and levels of ACh and choline (Ch) in whole mouse brain were examined 30 min following i.p. injection of 25 mg/kg of three tricyclic antidepressants (TAD's): amitriptyline, chlorimipramine, and imipramine. The effects of these agents on high affinity choline uptake (HACU), a rate-limiting, regulatory step in ACh synthesis, were also examined. All three TAD's inhibited ACh turnover (by 27-40%), increased Ch levels (by 33-37%), and inhibited HACU (IC50's from 1.7 - 6.8 X 10(-5) M). Two of the three drugs produced significant decreases in ACh levels. It is concluded that these agents possess anticholinergic activity which is independent of their previously demonstrated muscarinic receptor blocking capabilities. As substantial evidence has accumulated linking increased cholinergic function with depression, it is speculated that this additional anticholinergic activity may contribute to the clinical efficacy of the TAD's.