1. The concentrations of the oxidized and reduced substrates of the lactate-, beta-hydroxybutyrate- and glutamate-dehydrogenase systems were measured in rat livers freeze-clamped as soon as possible after death. The substrates of these dehydrogenases are likely to be in equilibrium with free NAD(+) and NADH, and the ratio of the free dinucleotides can be calculated from the measured concentrations of the substrates and the equilibrium constants (Holzer, Schultz & Lynen, 1956; Bücher & Klingenberg, 1958). The lactate-dehydrogenase system reflects the [NAD(+)]/[NADH] ratio in the cytoplasm, the beta-hydroxybutyrate dehydrogenase that in the mitochondrial cristae and the glutamate dehydrogenase that in the mitochondrial matrix. 2. The equilibrium constants of lactate dehydrogenase (EC 1.1.1.27), beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and malate dehydrogenase (EC 1.1.1.37) were redetermined for near-physiological conditions (38 degrees ; I0.25). 3. The mean [NAD(+)]/[NADH] ratio of rat-liver cytoplasm was calculated as 725 (pH7.0) in well-fed rats, 528 in starved rats and 208 in alloxan-diabetic rats. 4. The [NAD(+)]/[NADH] ratio for the mitochondrial matrix and cristae gave virtually identical values in the same metabolic state. This indicates that beta-hydroxybutyrate dehydrogenase and glutamate dehydrogenase share a common pool of dinucleotide. 5. The mean [NAD(+)]/[NADH] ratio within the liver mitochondria of well-fed rats was about 8. It fell to about 5 in starvation and rose to about 10 in alloxan-diabetes. 6. The [NAD(+)]/[NADH] ratios of cytoplasm and mitochondria are thus greatly different and do not necessarily move in parallel when the metabolic state of the liver changes. 7. The ratios found for the free dinucleotides differ greatly from those recorded for the total dinucleotides because much more NADH than NAD(+) is protein-bound. 8. The bearing of these findings on various problems, including the following, is discussed: the number of NAD(+)-NADH pools in liver cells; the applicability of the method to tissues other than liver; the transhydrogenase activity of glutamate dehydrogenase; the physiological significance of the difference of the redox states of mitochondria and cytoplasm; aspects of the regulation of the redox state of cell compartments; the steady-state concentration of mitochondrial oxaloacetate; the relations between the redox state of cell compartments and ketosis.

1. The recent recognition of the metabolic, as opposed to absorptive, functions of the small intestine prompted efforts the improve the preparation of metabolically competent columnar absorptive cells ('enterocytes') and to study their metabolic properties. 2. With this preparation, linear rates of O2 consumption are obtained for 40 min at 37 degrees C that are more than 50% higher than rates reported by other authors. 3. Among added substrates, glucose, glutamine and glutamate are the preferred fuels of respiration. The main nitrogenous products of glutamine metabolism are NH3, alanine and glutamate. Glutamine carbon was not detectable in citrulline or proline, in contrast with the findings of Windmueller & Spaeth [(1974) J. Biol. Chem. 249, 5070-5079] in the vascularly perfused small intestine. 4. The rates of O2 uptake in the presence of glutamine or glutamate are sufficient to account for the formation of the carbon skeleton of alanine from the amino acid substrate, i.e. the ratio of O2 used/alanine formed is greater than 1.5. 5. Added ADP and ATP are rapidly degraded to AMP and IMP to a large extent by release of hydrolytic enzymes from the enterocytes into the medium. 6. Chicken enterocytes isolated by the same method are more stable; linear rates of O2 uptake are maintained for 60-70 min.

This study was designed to identify the single-channel properties and molecular entity of ATP-sensitive K(+) (K(ATP)) channels in guinea pig gastric myocytes with patch-clamp recording and RT-PCR. Pinacidil and diazoxide activated K(ATP) currents in a glibenclamide-sensitive manner. The open probability of channels was enhanced by the application of 10 microM pinacidil from 0.085 +/- 0.04 to 0.20 +/- 0.05 (n = 7) and was completely blocked by 10 microM glibenclamide. Single-channel conductance was 37.3 +/- 2.5 pS (n = 4) between -80 and -20 mV in symmetrical K(+) gradient conditions. In inside-out mode, K(ATP) channels showed no spontaneous openings and were activated by the application of nucleotide diphosphates to the cytoplasmic side. These single-channel properties are similar to those of the nucleotide diphosphate-dependent K(+) channels in vascular smooth muscle, which are composed of Kir6.1 and sulfonylurea receptor (SUR)2B. RT-PCR demonstrated the presence of Kir6.1, Kir6.2, and SUR2B in guinea pig stomach smooth muscle cells. These results suggest that K(ATP) channels in smooth muscle cells of the guinea pig stomach are composed of Kir6.1 and SUR2B.

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This study tested the hypothesis that insulin-like growth factor I (IGF-I) expression is increased at sites of fibrosis in diseased intestine of patients with Crohn's disease (CD). IGF-I mRNA was quantified by RNase protection assay in uninvolved and involved intestine of 13 CD patients (10 ileum, 3 colon) and 7 ulcerative colitis (UC) patients (colon). In situ hybridization histochemistry compared the localization of IGF-I and procollagen alpha1(I) mRNAs. Masson's trichrome staining and immunohistochemistry for IGF-I precursor, alpha-smooth muscle actin (A), vimentin (V), desmin (D), and c-kit were used to examine the mesenchymal cell subtypes that express IGF-I and collagen in uninvolved and involved ileum and colon of CD patients and "normal" ileum and colon from noninflammatory controls. IGF-I mRNA was elevated in involved ileum and colon of patients with CD but not in involved colon of patients with UC. IGF-I and procollagen alpha1(I) mRNA showed overlapping distribution within fibrotic submucosa and muscularis propria of involved CD ileum and colon. In involved CD intestine, increased IGF-I precursor expression localized to mesenchymal cells in regions of tissue disorganization and fibrosis in muscularis mucosa, submucosa, and muscularis propria. In these regions, there were increased numbers of V(+) cells relative to normal or uninvolved intestine. Increased IGF-I expression was localized to cells with a phenotype typical of fibroblasts (V(+)/A(-)/D(-)), myofibroblasts (V(+)/A(+)/D(+)), and, to a lesser extent, cells with normal enteric smooth muscle phenotype (V(-)/A(+)/D(+)). We conclude that increased IGF-I expression in multiple mesenchymal cell subtypes and increased numbers of cells with fibroblast/myofibroblast phenotype are involved in fibrosis associated with CD.

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1. The concentration of HCO3- (independent of any change of pH) exerts different effects on glutamine metabolism in rat kidney-cortex tubules, hepatocytes and enterocytes.2. In kidney tubules HCO3- (10.5-50 MM) has no effect on glutaminase (EC 3.5.1.2), whereas glutamate dehydrogenase (EC 1.4.1.3) is inhibited as HCO3- concentration is increased. The result is that flux through the entire glutamate-to-glucose pathway is inhibited by increasing HCO3- concentrations. A large proportion (more than 30%) of the glutamine removed undergoes complete oxidation. 3. In hepatocytes, and to a smaller extent in enterocytes, HCO3- is an accelerator of glutaminase. Synthesis of glucose and urea from glutamine in hepatocytes increases as HCO3- concentration is increased. Calculations show that fumarate, formed via aspartate aminotransferase and arginino-succinate lyase, is the precursor of the glucose. There is no complete oxidation of the carbon skeleton of glutamine in hepatocytes. 4. Leucine at near-physiological concentrations (0.1-1 mM) is an accelerator of glutaminase in hepatocytes, but not in kidney tubules or in enterocytes. 5. The results are discussed in relation to regulation of acid/base balance in vivo.

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