Apoptosis, autophagy and ER stress in mevalonate cascade inhibition-induced cell death of human atrial fibroblasts.

3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) are cholesterol-lowering drugs that exert other cellular effects and underlie their beneficial health effects, including those associated with myocardial remodeling. We recently demonstrated that statins induces apoptosis and autophagy in human lung mesenchymal cells. Here, we extend our knowledge showing that statins simultaneously induces activation of the apoptosis, autophagy and the unfolded protein response (UPR) in primary human atrial fibroblasts (hATF). Thus we tested the degree to which coordination exists between signaling from mitochondria, endoplasmic reticulum and lysosomes during response to simvastatin exposure. Pharmacologic blockade of the activation of ER-dependent cysteine-dependent aspartate-directed protease (caspase)-4 and lysosomal cathepsin-B and -L significantly decreased simvastatin-induced cell death. Simvastatin altered total abundance and the mitochondrial fraction of proapoptotic and antiapoptotic proteins, while c-Jun N-terminal kinase/stress-activated protein kinase mediated effects on B-cell lymphoma 2 expression. Chemical inhibition of autophagy flux with bafilomycin-A1 augmented simvastatin-induced caspase activation, UPR and cell death. In mouse embryonic fibroblasts that are deficient in autophagy protein 5 and refractory to autophagy induction, caspase-7 and UPR were hyper-induced upon treatment with simvastatin. These data demonstrate that mevalonate cascade inhibition-induced death of hATF manifests from a complex mechanism involving co-regulation of apoptosis, autophagy and UPR. Furthermore, autophagy has a crucial role in determining the extent of ER stress, UPR and permissiveness of hATF to cell death induced by statins.

Latrunculin B increases force fluctuation-induced relengthening of ACh-contracted, isotonically shortened canine tracheal smooth muscle.

We hypothesized that differences in actin filament length could influence force fluctuation-induced relengthening (FFIR) of contracted airway smooth muscle and tested this hypothesis as follows. One-hundred micromolar ACh-stimulated canine tracheal smooth muscle (TSM) strips set at optimal reference length (Lref) were allowed to shorten against 32% maximal isometric force (Fmax) steady preload, after which force oscillations of +/-16% Fmax were superimposed. Strips relengthened during force oscillations. We measured hysteresivity and calculated FFIR as the difference between muscle length before and after 20-min imposed force oscillations. Strips were relaxed by ACh removal and treated for 1 h with 30 nM latrunculin B (sequesters G-actin and promotes depolymerization) or 500 nM jasplakinolide (stabilizes actin filaments and opposes depolymerization). A second isotonic contraction protocol was then performed; FFIR and hysteresivity were again measured. Latrunculin B increased FFIR by 92.2 +/- 27.6% Lref and hysteresivity by 31.8 +/- 13.5% vs. pretreatment values. In contrast, jasplakinolide had little influence on relengthening by itself; neither FFIR nor hysteresivity was significantly affected. However, when jasplakinolide-treated tissues were then incubated with latrunculin B in the continued presence of jasplakinolide for 1 more h and a third contraction protocol performed, latrunculin B no longer substantially enhanced TSM relengthening. In TSM treated with latrunculin B + jasplakinolide, FFIR increased by only 3.03 +/- 5.2% Lref and hysteresivity by 4.14 +/- 4.9% compared with its first (pre-jasplakinolide or latrunculin B) value. These results suggest that actin filament length, in part, determines the relengthening of contracted airway smooth muscle.

Immunogold evidence that neuronal gap junctions in adult rat brain and spinal cord contain connexin-36 but not connexin-32 or connexin-43.

Physiological and ultrastructural evidence indicates that gap junctions link many classes of neurons in mammalian central nervous system (CNS), allowing direct electrical and metabolic communication. Among at least six gap junction-forming connexin proteins in adult rat brain, connexin- (Cx) 32, Cx36, and Cx43 have been reported to occur in neurons. However, no connexin has been documented at ultrastructurally defined neuronal gap junctions. To address this question directly, freeze-fracture replica immunogold labeling (FRIL) and immunofluorescence (IF) were used to visualize the subcellular and regional localization of Cx36 in rat brain and spinal cord. Three antibodies were generated against different sequences in Cx36. By Western blotting, these antibodies detected protein at 36 and 66 kDa, corresponding to Cx36 monomer and dimer forms, respectively. After double-labeling for Cx36 and Cx43 by FRIL, neuronal gap junctions in inferior olive, spinal cord, and retina were consistently immunogold-labeled for Cx36, but none were labeled for Cx43. Conversely, Cx43 but not Cx36 was detected in astrocyte and ependymocyte gap junctions. In >250 Cx32/Cx43 single- and double-labeled replicas from 10 CNS regions, no neuronal gap junctions were labeled for either Cx32 or Cx43. Instead, Cx32 and Cx43 were restricted to glial gap junctions. By IF, Cx36 labeling was widely distributed in neuropil, including along dendritic processes and within neuronal somata. On the basis of FRIL identification of Cx36 in neuronal gap junctions and IF imaging of Cx36 throughout rat brain and spinal cord, neuronal gap junctions containing Cx36 appear to occur in sufficient density to provide widespread electrical and metabolic coupling in adult CNS.

Connexin30 in rodent, cat and human brain: selective expression in gray matter astrocytes, co-localization with connexin43 at gap junctions and late developmental appearance.

We previously presented evidence [Nagy et al. (1997) Neuroscience 78, 533-548] that, in addition to their ubiquitous expression of connexin43, astrocytes produce a second connexin suggested to be connexin30, a recently discovered member of the family of gap junction proteins. A connexin30 specific antibody was subsequently developed and utilized here to confirm and extend our earlier observations. On western blots, this antibody detected a 30,000 mol. wt protein in rat, mouse, cat and human brain, and exhibited no cross-reaction with connexin43, connexin26 or any other known connexins expressed in brain. Immunohistochemically, connexin30 was localized in astrocytes, at gap junctions between these cells and on the astrocyte side of gap junctions between astrocytes and oligodendrocytes. Double labelling revealed co-localization of connexin30 and connexin43 at astrocytic gap junctions. Punctate immunolabelling patterns for both connexins were qualitatively similar, but differences were evident. In contrast to regional connexin43 expression, diencephalic and hindbrain areas exhibited considerably greater expression than forebrain areas, subcortical perivascular astrocytic endfeet were more heavily labelled for connexin30, white matter tracts such as corpus callosum, internal capsule and anterior commissure were devoid of connexin30, and appreciable levels of connexin30 during development were not seen until about postnatal day 15. These results indicate that connexin30 is expressed by gray, but not white matter astrocytes, its distribution is highly heterogeneous in gray matter, it is co-localized with connexin43 at astrocytic gap junctions where it forms homotypic or heterotypic junctions, and its emergence is delayed until relatively late during brain maturation. Taken together, these results suggest that astrocytic connexin30 expression at both regional and cellular levels is subject to regulation in adult brain as well as during brain development.

Increased expression of cytosolic glutathione S-transferases in drug-resistant L5178Y murine lymphoblasts: chemical selectivity and molecular mechanisms.

The level of induction of three cytosolic glutathione-S-transferase (GST) classes has been compared in L5178Y murine lymphoblasts resistant to either the quinone-containing compound, hydrolyzed benzoquinone mustard (HBM), or the aromatic alkylating agent aniline mustard (AM). Three established cell lines, L5178Y/HBM2, L5178Y/HBM10, and the partial revertant, L5178Y/HBMR, were 2.5-, 6-, and 2.9-fold resistant to HBM and showed 3-, 11-, and 9-fold increases in GST activity, respectively, relative to the sensitive L5178Y cell line. Western blot analysis of cytosolic proteins showed overexpression of all three cytosolic GST classes pi, alpha, and mu, with predominance of the pi class. Northern blot analysis demonstrated corresponding elevations in the steady-state mRNA levels of each GST class. The level of GST-mu and -alpha isoforms correlated more closely with HBM resistance, whereas GST-pi, the predominant isoform in these cells, paralleled enzyme activity. These findings suggested that other factors such as quinone reductase may contribute to resistance. The AM-resistant cell line L5178Y/AM was 10-fold resistant to the alkylating agent AM, and GST activity was elevated 3.6-fold relative to the parental L5178Y cell line. Western blot analysis and Northern blot analysis provided evidence of overexpression of all three cytosolic GST classes but with marked predominance of the alpha class. These studies provide evidence that induction of GST isoforms in drug-resistant cells may have both a nonspecific as well as a selective component. The difference in isozyme profile between HBM- and AM-resistant cell lines emphasizes how structural differences, in particular, the nature of the electrophilic signal, may influence the pattern of induction of GST isozymes.