Despite abundant evidence for changes in mitochondrial membrane permeability in tumor necrosis factor (TNF)-mediated cell death, the role of plasma membrane ion channels in this process remains unclear. These studies examine the influence of TNF on ion channel opening and death in a model rat liver cell line (HTC). TNF (25 ng/ml) elicited a 2-and 5-fold increase in K ؉ and Cl ؊ currents, respectively, in HTC cells. These increases occurred within 5-10 min after TNF exposure and were inhibited either by K ؉ or Cl ؊ substitution or by K ؉ channel blockers (Ba 2؉ , quinine, 0.1 mM each) or Cl ؊ channel blockers (10 M 5-nitro-2-(3-phenylpropylamino)benzoic acid and 0.1 mM N-phenylanthranilic acid), respectively. TNF-mediated increases in K ؉ and Cl ؊ currents were each inhibited by intracellular Ca 2؉ chelation (5 mM EGTA), ATP depletion (4 units/ml apyrase), and the protein kinase C (PKC) inhibitors chelerythrine (10 M) or PKC 19 -36 peptide (1 M). In contrast, currents were not attenuated by the calmodulin kinase II 281-309 peptide (10 M), an inhibitor of calmodulin kinase II. In the presence of actinomycin D (1 M), each of the above ion channel blockers significantly delayed the progression to TNF-mediated cell death. Collectively, these data suggest that activation of K ؉ and Cl ؊ channels is an early response to TNF signaling and that channel opening is Ca 2؉ -and PKC-dependent. Our findings further suggest that K ؉ and Cl ؊ channels participate in pathways leading to TNF-mediated cell death and thus represent potential therapeutic targets to attenuate liver injury from TNF.

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Small conductance Ca2+-activated K+ (SK) channels have been cloned from mammalian brain, but little is known about the molecular characteristics of SK channels in nonexcitable tissues. Here, we report the isolation from rat liver of an isoform of SK3. The sequence of the rat liver isoform differs from rat brain SK3 in five amino acid residues in the NH3 terminus, where it more closely resembles human brain SK3. SK3 immunoreactivity was detectable in hepatocytes in rat liver and in HTC rat hepatoma cells. Human embryonic kidney (HEK-293) cells transfected with liver SK3 expressed 10 pS K+ channels that were Ca2+ dependent (EC(50) 630 nM) and were blocked by the SK channel inhibitor apamin (IC(50) 0.6 nM); whole cell SK3 currents inactivated at membrane potentials more positive than -40 mV. Notably, the Ca2+ dependence, apamin sensitivity, and voltage-dependent inactivation of SK3 are strikingly similar to the properties of hepatocellular and biliary epithelial SK channels evoked by metabolic stress. These observations raise the possibility that SK3 channels influence membrane K+ permeability in hepatobiliary cells during liver injury.

The growth characteristics and colonization potential of a transplantable melanoma administered to young (3 mo) and old (24 mo) C57BL/6 mice were investigated. After sc injection of B16-F10 melanoma cells, tumor growth was slower, and final tumor volume was less in the older mice. Furthermore, after iv injection of B16-F1 melanoma cells, the number of pulmonary colonies was also less, and the survival was greater in the older mice. These findings indicate an age advantage in this experimental tumor model that may be attributed to either physical or immunologic factors.

Swelling of hepatocytes and other epithelia activates volume-sensitive ion channels that facilitate fluid and electrolyte efflux to restore cell volume, but the responsible signaling pathways are incompletely defined. Previous work in model HTC rat hepatoma cells has indicated that swelling elicits ATP release, which stimulates P2 receptors and activates Cl ؊ channels, and that this mechanism is essential for hepatocellular volume recovery. , which is essential for ion channel activation and volume recovery, but that this increase does not stem from activation of volume-sensitive P2 receptors. Collectively, these observations imply that regulatory responses to hepatocellular swelling involve a dual requirement for a purinergic-independent Ca 2؉ signaling cascade and a Ca 2؉ -independent purinergic signaling pathway.Epithelia face substantial osmotic stresses from the vectorial transport of solutes that cause cell swelling and challenge cellular integrity. An adaptive response to cell swelling, termed regulatory volume decrease (RVD), 1 provides a dynamic safeguard against tissue injury produced by such stresses. RVD is mediated, in part, by the opening of swelling-activated K ϩ and Cl Ϫ channels in the plasma membrane, which leads to fluid and electrolyte efflux and consequent restoration of cell volume (1).Although it has been well appreciated that activation of both K ϩ and Cl Ϫ channels is critical for RVD, the mechanisms that couple cell swelling to ion channel activation and RVD exhibit tissue diversity.Among epithelia, hepatocytes are particularly susceptible to dynamic perturbations in cell volume, given the central role of the liver in nutrient uptake and metabolism (2). Whereas emerging evidence suggests that hepatocellular volume per se is an important determinant of several critical organ level functions, including glucose metabolism and bile formation (2), the mechanisms that govern hepatocellular volume regulation remain to be defined. We and others (3, 4) have provided evidence for the involvement of purinergic signaling in this process. In both HTC rat hepatoma cells and human hepatocytes, hypotonic swelling elicits ATP release, which stimulates P2 purinoreceptors, the activation of which leads to the opening of volume-sensitive Cl Ϫ channels (3, 4). This purinergic mechanism is essential for hepatocellular RVD. Although it is known that swelling-induced ATP release requires activation of phosphatidylinositol 3-kinase (5) and involves a putative member of the ATP-binding cassette family (6), the downstream effectors that couple osmosensitive P2 receptors to ion channel opening and RVD are unknown.In many cell types, including hepatocytes, activation of P2 receptors elicits increases in cytosolic [Ca 2ϩ ] ([Ca 2ϩ ] i ) through stimulation of phospholipase C, intracellular inositol trisphosphate (IP 3 ) formation, and activation of IP 3 receptors (7,8). However, P2 receptors have also been reported to be coupled to Ca 2ϩ -independent cellular effectors (9 -12). This leaves unresolved whether Ca 2ϩ...