The potassium channel Kv1.3 has recently been located to the inner mitochondrial membrane of lymphocytes. Here, we show that mouse and human cells that are genetically deficient in either Kv1.3 or transfected with siRNA to suppress Kv1.3-expression resisted apoptosis induced by several stimuli, including Bax over-expression. Retransfection of either Kv1.3 or a mitochondrial-targeted Kv1.3 restored cell death. Bax interacted with and functionally inhibited mitochondrial Kv1.3. Incubation of isolated Kv1.3-positive mitochondria with recombinant Bax, t-Bid, or toxins that bind to and inhibit Kv1.3 successively triggered hyperpolarization, formation of reactive oxygen species, release of cytochrome c, and marked depolarization. Kv1.3-deficient mitochondria were resistant to Bax, t-Bid, and the toxins. Mutation of Bax at K128, which corresponds to a conserved lysine in Kv1.3-inhibiting toxins, abrogated its effects on both Kv1.3 and mitochondria. These findings suggest that Bax mediates cytochrome c release and mitochondrial depolarization in lymphocytes, at least in part, via its interaction with mitochondrial Kv1.3

Many receptor systems use receptor clustering for transmembrane signaling. In this study, we show that acid sphingomyelinase (ASM) is essential for the clustering of CD40. Stimulation of lymphocytes via CD40 ligation results in ASM translocation from intracellular stores, most likely vesicles, into distinct membrane domains on the extracellular surface of the plasma membrane. Surface ASM initiates a release of extracellularly oriented ceramide, which in turn mediates CD40 clustering in sphingolipid-rich membrane domains. ASM, ceramide, and CD40 colocalize in the cap-like structure of stimulated cells. Deficiency of ASM, destruction of sphingolipid-rich rafts, or neutralization of surface ceramide prevents CD40 clustering and CD40-initiated cell signaling. These findings indicate that the ASM-mediated release of ceramide and/or metabolites of ceramide regulate clustering of CD40, which seems to be a prerequisite for cellular activation via CD40.

The margatoxin-sensitive Kv1.3 is the major potassium channel in the plasma membrane of T lymphocytes. Electron microscopy, patch clamp, and immunological studies identified the potassium channel Kv1.3, thought to be localized exclusively in the cell membrane, in the inner mitochondrial membrane of T lymphocytes. Patch clamp of mitoplasts and mitochondrial membrane potential measurements disclose the functional expression of a mitochondrial margatoxin-sensitive potassium channel. To identify unambiguously the mitochondrial localization of Kv1.3, we employed a genetic model and stably transfected CTLL-2 cells, which are genetically deficient for this channel, with Kv1.3. Mitochondria isolated from Kv1.3-reconstituted CTLL-2 expressed the channel protein and displayed an activity, which was identical to that observed in Jurkat mitochondria, whereas mitochondria of mock-transfected cells lacked a channel with the characteristics of Kv1.3. Our data provide the first molecular identification of a mitochondrial potassium conductance.

SummaryHaem-oxygenase-1 (HO-1) has been shown to exert anti-inflammatory, antiapoptotic and anti-proliferative effects. We investigated HO-1 expression in patients with inflammatory bowel disease (

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General aspects of apoptosisProgrammed cell death or apoptosis is an evolutionaryconserved mechanism employing a complex signalling machinery for the removal of cells. Removal of cells is important, for example, during development, organ homeostasis, and elimination of auto-reactive lymphocytes and neoplastic, damaged or infected cells.In general, stimuli triggering apoptosis can be separated into two groups, i.e. physiological and stress stimuli. Physiological stimuli include surface receptors such as TNF or CD95; examples of stress stimuli are UV light, irradiation, viral and bacterial infections, etc. While many physiological stimuli trigger death via surface receptors, the initiation of stress-induced apoptosis is much less defined, but seems to involve mitochondria at a very early stage of the intracellular signalling cascade.Receptor-induced apoptosis CD95 initiates apoptosis (Fig. 1) by a translocation of the acid sphingomyelinase from an intracellular compartment onto the cell membrane surface (Grassmé et al. 2001a,b). The cell membrane contains small distinct domains that are mainly composed of sphingolipids and cholesterol (Simons & Ikonen, 1997). Homo-and heterotypic interactions of sphingolipids and cholesterol render these domains less fluid than the other parts of the cell membrane and result in the separation of these small domains from phospholipids in the cell membrane (Simons & Ikonen, 1997). These domains that are also named rafts are transformed by acid sphingomyelinasereleased ceramide: ceramide triggers a fusion of many small raft domains to a large platform (Grassmé et al. 2001a,b) that serves to cluster CD95, an event required for the initiation of specific CD95 signalling (Grassmé et al. 2001a). Clustered CD95 efficiently recruits an adapter protein, Fas-associated death domain (FADD), that binds to a protease, caspase 8 (Boldin et al. 1996;Muzio et al. 1996). Caspases are a family of cysteine proteases that cleave their substrates specifically after aspartate residues (for review see Green & Kroemer, 1998). They are synthesized as inactive pro-enzymes that are converted to catalytically active proteases by limited proteolysis (Green & Kroemer, 1998). Caspase 8 transfers the apoptotic signal from CD95 via Bcl-2-like proteins (see below) and unknown mechanisms to mitochondria or directly to further caspases, in particular caspase 3. This caspase serves to execute apoptosis by cleavage of a large number of intracellular proteins, including enzymes involved in genome function, regulators of cell-cycle progression and structural proteins of the nucleus and cytoskeleton (Green & Kroemer, 1998). Cleavage of these proteins results in morphological alterations indicative of apoptosis, e.g. membrane-blebbing and DNA-fragmentation (Green & Kroemer, 1998). In summary, these receptor-initiated signalling pathways seem to employ mitochondria primarily to enhance the apoptotic signal. Special Review Series -Biogenesis and Physiological Adaptation of Mitochondria Role of mitochondria in apoptosisErich...

Receptor clustering is a key event in the initiation of signaling by many types of receptor molecules. Here, we provide evidence for the novel concept that clustering of a ligand is a prerequisite for clustering of the cognate receptor. We show that clustering of the CD40 receptor depends on reciprocal clustering of the CD40 ligand (gp39, CD154). Clustering of the CD40 ligand is mediated by an association of the ligand with p53, a translocation of acid sphingomyelinase (ASM) to the cell membrane, an activation of the ASM, and a formation of ceramide. Ceramide appears to modify preexisting sphingolipidrich membrane microdomains to fuse and form ceramide-enriched signaling platforms that serve to cluster CD40 ligand. Genetic deficiency of p53 or ASM or disruption of ceramide-enriched membrane domains prevents clustering of CD40 ligand. The functional significance of CD40 ligand clustering is indicated by the finding that clustering of CD40 on B lymphocytes upon co-incubation with CD40 ligand-expressing T cells depends on clustering of the CD40 ligand and is abrogated by inhibition of CD40 ligand clustering.Receptor clustering or aggregation is a central event in the signaling of many types of receptor molecules and is initiated by the interaction of a ligand with its cognate receptor. If the ligand is membrane-bound, the contact site between the ligand and the receptor resembles some features of a neurological synapse and, thus, has been also named immune synapse (1).Many receptors appear to aggregate in sphingolipid-rich membrane rafts, which contain a high concentration of sphingolipids and cholesterol (2-4). The biophysical properties of these membrane domains cause them to separate from the phospholipids in the cell membrane and to resist breakdown by some detergents (2-4). Therefore, these small rafts were also named detergent-insensitive, glycosphingolipid-rich domains. Most studies on receptor clustering focused on the role of intracellular signaling molecules (for recent reviews, see Refs. 5 and 6); however, we have recently suggested that membrane changes also contribute to receptor clustering (7-10). These studies revealed that stimulation of CD95 or CD40 triggers the fusion of acid sphingomyelinase (ASM) 1 -containing, intracellular vesicles with the cell membrane (7, 9, 10). This fusion results in the exposure of the enzyme to the extracellular leaflet of the cell membrane. ASM activity results in the formation of extracellularly oriented ceramide from sphingomyelin (7, 9, 10). Surface ceramide seems to reorganize small lipid rafts into larger platforms, which serve to trap and cluster CD95 or CD40, respectively (7-10).Although the central role of receptor clustering for signaling is widely accepted and although many intracellular signaling mechanisms of the clustering process have been identified, the role of the ligand in the clustering of its cognate receptor is unknown. To test the hypothesis that clustering of a ligand is involved in the clustering of its receptor, we examined the CD40-CD40 ligand...

Aims To investigate the in¯uence of combined ritonavir (RTV) and saquinavir (soft-gelatin capsule formulation; SQV) on systemic exposure to SQV with a view to optimizing the dosing regimen of combined RTV and SQV antiretroviral therapy.Methods In this open labelled, randomized, parallel group study, SQV and RTV were administered twice daily for 14 days to groups of eight healthy subjects. The two antiretrovirals were either administered alone (800 mg SQV, regimen A, and 400 mg RTV, B) or in combination at various dose levels (RTV : SQV: 400 : 400 mg, C; 300 : 600 mg, D; 200 : 800 mg, E; 300 : 800 mg, F; 400 : 800 mg, G; and 400 : 600 mg, H). Pharmacokinetic parameters of saquinavir and ritonavir were determined and adverse events, vital signs, and clinical laboratory variables recorded. Results RTV substantially increased the plasma concentration of saquinavir for all dose combinations, compared with SQV alone. Based on the primary statistical analysis there was an overall 17-, 22-, and 23-fold increase in saquinavir AUC(0,24 h) on day 14 with regimens E, F, and G, respectively (with con®dence intervals of 10±30, 13±37, and 13±39). The lowest combination dose of RTV (200 : 800 mg; E) signi®cantly increased the saquinavir AUC(0,24 h) from below 5 to 57 mg ml x1 h, which was higher than the exposure obtained with the 400 : 400 mg twice daily regimen (i.e. 36 mg ml x1 h). RTV also reduced intersubject variability in AUC(0,24 h) for saquinavir from 105% to 32±68%, and C max (0,24 h) from 124% to 30±49%. In contrast, SQV showed no clinically signi®cant effect on the pharmacokinetics of ritonavir. The combination regimens were well tolerated, with the least number of adverse events recorded for the 200 : 800 mg (RTV : SQV) combination regimen. Conclusions RTV signi®cantly increases saquinavir exposure as a consequence of inhibiting SQV metabolism and possibly P-glycoprotein ef¯ux. Pharmacokinetic and safety pro®les obtained in the current study indicate that the use of a combination with a lower dose of RTV and a higher dose of SQV than the 400 : 400 mg combination frequently used in clinical practice should be further explored.