Multiorgan apoptosis occurs during sepsis. Following cecal ligation and puncture (CLP) in rats, thymocytes underwent apoptosis in a time-dependent manner. C5a blockade dramatically reduced thymocyte apoptosis as measured by thymic weight, binding of annexin V to thymocytes, and laddering of thymocyte DNA. When C5a was generated in vivo by infusion of purified cobra venom factor (CVF), thymocyte apoptosis was significantly increased. Similar results were found when CVF was injected in vivo during the early stages of CLP. In animals 12 hours after induction of CLP, there was an increase in the activities of caspase-3, -6, and -9, but not caspase-1 and -8. Cytosolic cytochrome c levels increased by twofold, whereas mitochondrial levels showed a 50% decrease. Western blot analysis revealed that the content of Bcl-X L (but not of Bcl-2, BAX, Bad, and Bim) significantly decreased in thymocytes after CLP. C5a blockade in the sepsis model almost completely inhibited caspase-3, -6, and -9 activation, significantly preserved cytochrome c in the mitochondrial fraction, and restored Bcl-X L expression. These data suggest that systemic activation of complement induces C5a-dependent apoptosis of thymocytes and that the blockade of C5a during sepsis rescues thymocytes from apoptosis.

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splicing generates diverse transcripts. Am. J. Physiol. 274 (Cell Physiol. 43): C1215-C1225, 1998.-Na ϩ -myo-inositol cotransport activity generally maintains millimolar intracellular concentrations of myo-inositol and specifically promotes transepithelial myoinositol transport in kidney, intestine, retina, and choroid plexus. Glucose-induced, tissue-specific myo-inositol depletion and impaired Na ϩ -myo-inositol cotransport activity are implicated in the pathogenesis of diabetic complications, a process modeled in vitro in cultured human retinal pigment epithelium (RPE) cells. To explore this process at the molecular level, a human RPE cDNA library was screened with a canine Na ϩ -dependent myo-inositol cotransporter (SMIT) cDNA. Overlapping cDNAs spanning 3569 nt were cloned. The resulting cDNA sequence contained a 2154-nt open reading frame, 97% identical to the canine SMIT amino acid sequence. Genomic clones containing SMIT exons suggested that the cDNA is derived from at least five exons. Hypertonic stress induced a time-dependent increase, initially in a 16-kb transcript and subsequently in 11.5-, 9.8-, 8.5-, 3.8-, and ϳ1.2-kb SMIT transcripts, that was ascribed to alternate exon splicing using exon-specific probes and direct cDNA sequencing. The human SMIT gene is a complex multiexon transcriptional unit that by alternate exon splicing generates multiple SMIT transcripts that accumulate differentially in response to hypertonic stress. human retinal pigment epithelial cells; hypertonic stress; exon splicing; osmoregulation; diabetes mellitus THE WATER-SOLUBLE CYCLIC hexitol myo-inositol is a constituent of virtually every living cell and an essential nutrient for most mammalian cells in culture. Intracellular concentrations 50-to 1,000-fold greater than that of extracellular fluid are with few exceptions (25) ascribed to the action of membrane-associated, Na ϩ -dependent myo-inositol cotransporters (SMITs) (2). At the cellular level, myo-inositol is an obligate and sometimes rate-limiting (19) substrate for phosphoinositide synthesis. Along with sorbitol, taurine, and betaine, myo-inositol belongs to a family of alternative nonionic organic intracellular osmolytes whose regulated accumulation and efflux in response to hypertonic stress preserve intracellular volume and tonicity without perturbing the ionic milieu (2, 5). Organic osmolyte accumulation is coordinated by the regulated expression and/or activity of osmotically responsive, osmolytespecific genes or gene products: distinct Na ϩ -cotransporters for myo-inositol, taurine, and betaine and aldose reductase (AR) for the synthesis of sorbitol from glucose. Gradient-dependent organic osmolyte efflux is thought to be regulated primarily but not exclusively by a nonselective, ATP-dependent, volume-sensitive organic anion channel (2,14,27). Thus, at any given tonicity, the relative abundance of each osmolyte depends in part on substrate availability, the abundance and activity of their transporter or biosynthetic enzyme, and their efflux or degradation. T...

myo-Inositol is a ubiquitous intracellular organic osmolyte and phosphoinositide precursor maintained at millimolar intracellular concentrations through the action of membrane-associated Na+- myo-inositol cotransporters (SMIT). Functional cloning and expression of a canine SMIT cDNA, which conferred SMIT activity in Xenopus oocytes, predicted a 718-amino acid peptide homologous to the Na+-glucose cotransporter with a potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites. A consistent ∼1.0- to 13.5-kb array of transcripts hybridizing with this cDNA are osmotically induced in a variety of mammalian cells and species, yet SMIT activity appears to vary among different tissues and species. An open reading frame on human chromosome 21 (SLC5A3) homologous to that of the canine cDNA (96.5%) is thought to comprise an intronless human SMIT gene. Recently, this laboratory ascribed multiply sized, osmotically induced SMIT transcripts in human retinal pigment epithelial cells to the alternate utilization of several 3′-untranslated SMIT exons. This article describes an alternate splice donor site within the coding region that extends the open reading frame into the otherwise untranslated 3′ exons, potentially generating novel SMIT isoforms. In these isoforms, the last putative transmembrane domain is replaced with intracellular carboxy termini containing a novel potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites, and this could explain the heterogeneity in the regulation and structure of the SMIT.