Nucleosomes inhibit phagocytosis of apoptotic thymocytes by peritoneal macrophages from MRL+/+ lupus-prone mice

The nucleosome, the basic structure of chromatin and normal product of cell apoptosis, plays a pivotal role both in the induction and the pathogenesis of systemic lupus erythematosus (SLE). Nucleosomes have been found to circulate at high levels in patients with SLE and apoptosis of lymphoid cells is increased during human and murine lupus. In this study, we examined the presence of possible defects in clearance mechanisms of apoptotic cells in murine lupus, and questioned further whether nucleosomes could compromise this phagocytic process. There did not appear to be any intrinsic functional defect of macrophages from young MRL+/+ lupus-prone mice to recognize and phagocytose apoptotic thymocytes. Nucleosomes, as a mimic of increased cell apoptotsis in vivo, induced a strong, dose-dependent, inhibition of phagocytosis of apoptotic thymocytes by young, pre-autoimmune, macrophages of MRL+/+ mice, whereas macrophages of non-autoimmune C3H mice only exhibited a trend to inhibition. The nucleosome-elicited inhibitory effect persisted during the development of the autoimmune response and appeared to be specific for the molecular mechanisms involved in macrophage phagocytosis of apoptotic cells. Our data suggest that nucleosome elicited inhibition of phagocytosis of apoptotic cells by MRL+/+ macrophages before the onset of the autoimmune response contribute, in a positive loop, to sustain and/or augment the levels of circulating (and potentially immunogenic) nucleosomes in lupus.     

Ubiquitination of histone H3 in elongating spermatids of rat testes

Because of the potential role of histone ubiquitination in altering chromatin structure, we characterized the levels of ubiquitination of specific histones in meiotic and postmeiotic germ cells in rat testes by two-dimensional gel electrophoresis. The levels of the major ubiquitinated histone forms, mono- and poly-ubiquitinated H2A, were highest in the pachytene spermatocyte stage, declined thereafter through the round spermatid stage, and reached their lowest levels in elongating spermatids. Three additional ubiquitinated histone species, besides H2A, were detected using anti-ubiquitin antibodies specifically in the fraction enriched in elongating spermatids. Based on their electrophoretic mobilities, they corresponded to uH3, uTH3, and uH2B. Polyubiquitinated forms of these proteins were also observed. The identity of these proteins was confirmed by immunoblotting with anti-H3 antisera and by differential extraction of the proteins from the nucleus with increasing salt concentrations. This is the first report of ubiquitination of H3 in vivo. We speculate that its ubiquitination could loosen the nucleosome structure in preparation for histone removal, be a consequence of nucleosome relaxation or disruption caused by other means, or target H3 for degradation.     

SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl]-1H- imidazole hydrochloride) stimulates phosphoinositide hydrolysis in human U373 MG astrocytoma cells

SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl]-1H-imi dazole hydrochloride) stimulated the accumulation of [3H]inositol monophosphates ([3H]IP1) in human U373 MG astrocytoma cells prelabelled with [3H]inositol (EC50 15 +/- 1 microM, Hill coefficient 3.8 +/- 0.4). SK&F 96365-induced accumulation of [3H]IP1 increased linearly with time, but there was no initial rapid formation of [3H]IP3. SK&F 96365 also stimulated [3H]IP1 accumulation in human HeLa cells, but only to a small extent in slices of rat cerebral cortex and guinea-pig cerebellum. SK&F 96365-induced accumulation of [3H]IP1 in U373 MG cells increased as extracellular Ca2+ was increased from nominally zero to 4 mM, but there was no evidence that SK&F 96365 induced any marked entry of Ca2+ into cells; only an inhibition of store-refilling-induced Ca2+ entry was apparent. Further, the response to SK&F 96365 was additive with that to the Ca2+ ionophore ionomycin. Depolarization of the cells with raised K+ produced only a small stimulation of phosphoinositide hydrolysis. SK&F 96365 caused the release of Ca2+ from intracellular stores in U373 MG cells (EC50 26 +/- 14 microM), but thapsigargin induced only a small accumulation of [3H]IP1. Miconazole, another N-substituted imidazole, also stimulated [3H]IP1 accumulation in U373 cells.     

Archaeal nucleosomes

Archaea contain histones that have primary sequences in common with eukaryal nucleosome core histones and a three-dimensional structure that is essentially only the histone fold. Here we report the results of experiments that document that archaeal histones compact DNA in vivo into structures similar to the structure formed by the histone (H3+H4)2 tetramer at the center of the eukaryal nucleosome. After formaldehyde cross-linking in vivo, these archaeal nucleosomes have been isolated from Methanobacterium thermoautotrophicum and Methanothermus fervidus, visualized by electron microscopy on plasmid and genomic DNAs, and shown by immunogold labeling, SDS/PAGE, and immunoblotting to contain archaeal histones, cross-linked into tetramers. Archaeal nucleosomes protect approximately 60 bp of DNA and multiples of approximately 60 bp from micrococcal nuclease digestion, and immunoprecipitation has demonstrated that most, but not all, M. fervidus genomic DNA sequences are associated in vivo with archaeal histones.     

Differential acetylcholine and GABA release from cultured chick retina cells

In the present work we investigated the mechanisms controlling the release of acetylcholine (ACh) and of gamma-aminobutyric acid (GABA) from cultures of amacrine-like neurons, containing a subpopulation of cells which are simultaneously GABAergic and cholinergic. We found that 81.2 +/- 2.8% of the cells present in the culture were stained immunocytochemically with an antibody against choline acetyltransferase, and 38.5 +/- 4.8% of the cells were stained with an antibody against GABA. Most of the cells containing GABA (87.0 +/- 2.9%) were cholinergic. The release of acetylcholine and GABA was mostly Ca2+-dependent, although a significant release of [3H]GABA occurred by reversal of its transporter. Potassium evoked the Ca2+-dependent release of [3H]GABA and [3H]acetylcholine, with EC50 of 31.0 +/- 1.0 mm and 21.6 +/- 1.1 mm, respectively. The Ca2+-dependent release of [3H]acetylcholine was significantly inhibited by 1 micrometer tetrodotoxin and by low (30 nm) omega-conotoxin GVIA (omega-CgTx GVIA) concentrations, or by high (300 nm) nitrendipine (Nit) concentrations. On the contrary, the release of [14C]GABA was reduced by 30 nm nitrendipine, or by 500 nm omega-CgTx GVIA, but not by this toxin at 30 nm. The release of either transmitters was unaffected by 200 nm omega-Agatoxin IVA (omega-Aga IVA), a toxin that blocks P/Q-type voltage-sensitive Ca2+ channels (VSCC). The results show that Ca2+-influx through omega-CgTx GVIA-sensitive N-type VSCC and through Nit-sensitive L-type VSCC induce the release of ACh and GABA. However, the significant differences observed regarding the Ca2+ channels involved in the release of each neurotransmitter suggest that in amacrine-like neurons containing simultaneously GABA and acetylcholine the two neurotransmitters may be released in distinct regions of the cells, endowed with different populations of VSCC.     

A high affinity binding site for [3H]-Dofetilide on human leukocytes

Certain Class III anti-arrhythmic agents have been shown to interact with human leukocytes and after antigenic and mitogenic activation. We hypothesized that a binding site for the Class III anti-arrhythmic agent, dofetilide, would exist on human leukocytes. Analysis of binding isotherms defined the presence of a single high affinity binding site on mononuclear cells and neutrophils: Kd 26+/-4 nm, Bmax 61+/-14 fmol/10( 6) cells and Kd 33+/-14 nm, Bmax 163+/-45 fmol/10(6) cells, respectively. Other Class III drugs inhibited [3H]-dofetilide binding at physiologically relevant concentrations, but the IC50 values of E4031 and quinidine were significantly higher for leukocytes than for cardiac myocytes. Interestingly, verapamil inhibited [3H]-dofetilide binding to leukocytes, but not to cardiac myocytes at physiologic concentrations (10 microM). Charybdotoxin and tetraethlyammonium inhibited [3H]-dofetilide binding to leukocytes at microM mm concentrations, respectively, however, apamin did not inhibit binding even at 1 microM concentrations. These data suggest that a Ca2+-activated K+ channel, like K(Ca) mini (apamin-insensitive isoform), is a candidate for the leukocyte [3H]-dofetilide binding site. To assess the functional significance of defetilide binding to leukocyte biology, we evaluated fMLP-stimulated superoxide production in the presence or absence of dofetilide. Dofetilide, at 30 nm suppressed of superoxide production. In conclusion, dofetilide binds to human leukocytes at physiologic concentrations and this binding alters leukocyte function possibly through interaction with a Ca2+-activated K+ channel.     

The deletion of six ORFs of unknown function from Saccharomyces cerevisiae chromosome VII reveals two essential genes: YGR195w and YGR198w

Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod outer segments (ROS) and its properties determined. The enzyme activity was assayed using either a sonicated microdispersion of 1,2-diacyl-sn-[2(3)H]glycerol-3-phosphocholine (PC), or [14C]ethanol. Using [3H]PC and ethanol as a substrate, we were able to detect the hydrolytic properties as well as the transphosphatidylation reaction catalyzed by phospholipase D (PLD): formation of [3H]phosphatidic acid and phosphatidylethanol [3H]PtdEt; whereas with [14C]ethanol or [3H]glycerol in the absence of exogenous PC, only transphosphatidylation reactions were detected (formation of [14C]PtdEt or [3H]phosphatidylglycerol, respectively). The use of varying concentrations of [3H]PC and 400 mM of ethanol gave an apparent Km value for PC of 0.51 mM and a Vmax value of 111 nmol x h(-1) x (mg protein)(-1). The activity was linear up to 60 min of incubation and up to 0.2 mg of protein. The optimal ethanol concentration was determined to be 400 mM, with an apparent Km of 202 mM and a Vmax value for ethanol of 125 nmol x h(-1) x (mg protein)(-1). A clear pH optimum was observed around 7. PLD activity was increased in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate or sodium deoxycholate and inhibited with Triton X-100. The enzyme activity was also activated in the presence of Ca2+ or Mg2+ (1 mM) although these ions were not required for measuring PLD activity. The high specific activity of PLD found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to ROS. The present report is the first evidence of PLD activity associated with photoreceptor ROS.     

Nuclear muscarinic acetylcholine receptors in corneal cells from rabbit

PURPOSE: Previous studies have indicated that muscarinic acetylcholine receptors (mAChR) may be present in an unexpected, unique location and play a singular role in cellular growth regulation of rabbit corneal epithelium that may be of general physiologic significance if found in other cells. The purpose of this study was to examine rabbit corneas and corneal cells in culture to determine mAChR location and tissue distribution. METHODS: Using [3H]-propylbenzilylcholine mustard ([3H]PrBChM), which binds covalently to the active site of mAChR, rabbit corneal cross-sections, cultured corneal keratocytes, epithelial and endothelial cells, as well as nuclei isolated from these cultured corneal cells were labeled, stained, and autoradiographed. Nuclei labeled with [3H]PrBChM were further analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. RESULTS: Direct visual confirmation of the localization of mAChRs was obtained. MAChR were found in epithelial and endothelial layers of fresh-frozen corneal cross-sections, in cultured rabbit epithelial and endothelial cells, and on isolated rabbit epithelial and endothelial cell nuclei. mAChR were not detectable in keratocytes with these techniques. When [3H]PrBChM-labeled nuclei from cultured corneal cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, epithelial and endothelial samples showed specific mAChR binding, whereas binding to keratocyte nuclei was not detectable. CONCLUSIONS: As a result of these findings, a revised hypothesis is suggested for the locations and possible functions of mAChR in regulation of growth in corneal and other cells.     

The endotoxin of "brucellae"

Hen oviduct chromatin was digested with DNAase II and two fractions were isolated: MgCl2-insoluble chromatin and MgCl2-soluble chromatin. The former contained 14 and 50% of the total DNA after a digestion time of 3 and 30 min, respectively. The fraction was characterized in sucrose gradients by a peak sedimenting at 11S. In the course of DNAase digestion this fraction lost most of its estrogen receptors as assayed by [3H]estradiol exchange reaction. The specific radioactivity of chromatin was particularly low in the 11S region. The MgCl2-soluble chromatin contained at most 5.1% of the total DNA. In sucrose gradients the fraction displayed peaks at 4S and 14S. After a 30 min DNAase digestion the specific radioactivity of chromatin in this fraction exceeded that of the MgCl2-insoluble fraction 7.7 fold. Material sedimenting at 14 S and at larger S values was enriched in estrogen receptors. The results suggest that estrogen receptors are unevenly distributed on hen oviduct chromain.     

Purification and characterization of a Mg2+-dependent endonuclease (AN34) from etoposide-treated human leukemia HL-60 cells undergoing apoptosis

An important biochemical hallmark of apoptosis is the cleavage of chromatin into oligonucleosomal fragments. Here, we purified a Mg2+-dependent endonuclease from etoposide-treated HL-60 cells undergoing apoptosis. High levels of Mg2+-dependent endonuclease activity were detected in etoposide-treated HL-60 cells, and this activity increased in a time-dependent manner following etoposide treatment. Such an activity could not be detected in untreated cells or in cells treated with etoposide in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD-fmk) or the serine protease inhibitor tosyl-L-phenylalanine chloromethyl ketone (TPCK). This Mg2+-dependent endonuclease was purified by a series of chromatographic procedures. The enzyme preparation showed a single major protein band with Mr 34,000, determined by SDS-PAGE. The presence of the Mr 34,000 Mg2+-dependent endonuclease was also confirmed by activity gel analysis. The enzyme required only Mg2+ for full activity. pH optimum was in the range of 6.5-7.5. This enzyme introduced single- and double-strand breaks into SV40 DNA and produced internucleosomal DNA cleavage in isolated nuclei from untreated cells. The DNA breaks were terminated with 3'-OH, consistent with characteristic products of apoptotic chromatin fragmentation. We propose to designate this Mr 34,000 Mg2+-dependent endonuclease AN34 (apoptotic nuclease Mr 34,000).     

Fragmentation of chromatin with 125I radioactive disintegrations

The DNA in Chinese hamster cells was labeled first for 3 h with [3H]TdR and then for 3 h with [125I]UdR. Chromatin was extracted, frozen, and stored at -30 degrees C until 1.0 X 10(17) and 1.25 X 10(17) disintegrations/g of labeled DNA occurred for 125I and 3H respectively. Velocity sedimentation of chromatin (DNA with associated chromosomal proteins) in neutral sucrose gradients indicated that the localized energy from the 125I disintegrations, which gave about 1 double-strand break/disintegration plus an additional 1.3 single strand breaks, selectively fragmented the [125I] chromatin into pieces smaller than the [3H] chromatin. In other words, 125I disintegrations caused much more localized damage in the chromatin labeled with 125I than in the chromatin labeled with 3H, and fragments induced in DNA by 125I disintegrations were not held together by the associated chromosomal proteins. Use of this 125I technique for studying chromosomal proteins associated with different regions in the cellular DNA is discussed. For these studies, the number of disintegrations required for fragmenting DNA molecules of different sizes is illustrated.     

Specific transfer of 3H from D-[3-3H]gluconic acid into L-tartaric acid in vitaceous plants

Transfer of 3H from D-gluconic acid, specifically labelled with 3H at C-2 or C-3 and 14C at C-1, C-2, or C-3, 4, to L(+)-tartaric acid was examined in leaves and berries of Vitis labrusca cv Delaware and in leaves of Parthenocissus quinquefolia. 3H located at C-3 of D-gluconic acid was highly conserved in this transfer, yielding a 3H/14C ratio between 3.3 and 14 in the light and between 11 and 22 in the dark. These experiments strongly suggest that a portion of the 3H present in L(+)-tartaric acid may have been transferred from D-gluconic acid to L(+)-tartaric acid, possibly via NADP[3H] through a redox process involving reduction of L-xylo-2-hexulosonate (2-keto-L-idonate). Both [3H]-tartaric acid and [14C]tartaric acid synthesized in grape leaves from D-[3-3H, 2-14C]gluconic acid, or [3-3H, 3,4-14C]gluconic acid were characterized as L(+)-chiral form exclusively, the naturally occurring from of tartaric acid.     

Resistance of A-431 epidermoid carcinoma cells to early membrane changes induced by tumour promoters

The A-431 human epidermoid carcinoma cell line was resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibition of epidermal growth factor binding, enhanced incorporation of [3H]choline into phospholipids and uptake of 86Rb an [3H]2-deoxyglucose. The cells were also resistant to TPA-stimulated release of radioactive choline derivatives and arachidonic acid from cells prelabelled with [3H]choline or [14C]arachidonic acid, respectively. The A-431 cells did not metabolise [3H]TPA. Despite their TPA-unresponsiveness, A-431 cells contained specific, high affinity binding sites for [3H]phorbol-12,13-dibutyrate with characteristics similar to other cultured cell lines.     

Yeast L double-stranded ribonucleic acid is synthesized during the G1 phase but not the S phase of the cell cycle

The cytoplasm of Saccharomyces cerevisiae contains two major classes of protein-encapsulated double-stranded ribonucleic acids (dsRNA's), L and M. Replication of L and M dsRNA's was examined in cells arrested in the G1 phase by either alpha-factor, a yeast mating pheromone, or the restrictive temperature for a cell cycle mutant (cdc7). [3H]uracil was added during the arrest periods to cells prelabeled with [14C]uracil, and replication was monitored by determining the ratio of 3H/14C for purified dsRNA's. Like mitochondrial deoxyribonucleic acid, both L and M dsRNA's were synthesized in the G1 arrested cells. The replication of L dsRNA was also examined during the S phase, using cells synchronized in two different ways. Cells containing the cdc7 mutation, treated sequentially with alpha-factor and then the restrictive temperature, enter a synchronous S phase when transferred to permissive temperature. When cells entered the S phase, synthesis of L dsRNA ceased, and little or no synthesis was detected throughout the S phase. Synthesis of L dsRNA was also observed in G1 phase cells isolated from asynchronous cultures by velocity centrifugation. Again, synthesis ceased when cells entered the S phase. These results indicate that L dsRNA replication is under cell cycle control. The control differs from that of mitochondrial deoxyribonucleic acid, which replicates in all phases of the cell cycle, and from that of 2-micron DNA, a multiple-copy plasmid whose replication is confined to the S phase.     

Calcium-dependent [3H]acetylcholine release and muscarinic autoreceptors in rat cortical synaptosomes during development

A number of presynaptic cholinergic parameters (high affinity [3H]choline uptake, [3H]acetylcholine synthesis, [3H]acetylcholine release, and autoinhibition of [3H]acetylcholine release mediated by muscarinic autoreceptors) were comparatively analyzed in rat brain cortex synaptosomes during postnatal development. These various functions showed a differential time course during development. At 10 days of age the release of [3H]acetylcholine evoked by 15 mM KCl from superfused synaptosomes was Ca2+-dependent but insensitive to the inhibitory action of extrasynaptosomal acetylcholine. The muscarinic autoreceptors regulating acetylcholine release were clearly detectable only at 14 days, indicating that their appearance may represent a criterion of synaptic maturation more valuable than the onset of a Ca2+-dependent release.     

Identification of a rat testis-specific gene encoding a potential rat outer dense fibre protein

Histamine H2-receptor antagonistic properties of the anti-ulcer agent T-593, (+/-)-(E)-1-[2-hydroxy-2-(4-hydroxyphenyl)ethyl] -3-[2[[[5-(methylamino)methyl-2-furyl]methyl]thio] ethyl] -2-(methylsulfonyl)guanidine, were investigated on [14C]aminopyrine accumulation in isolated canine gastric mucosal cells and compared with those of ranitidine and famotidine. The potency of T-593-inhibition of [14C]aminopyrine accumulation stimulated by 10(-4) M histamine, with an IC50 value of 1.85 x 10(-6) M, was approximately 5 times greater than that of ranitidine, but half that of famotidine. T-593 did not affect [14C]aminopyrine accumulation stimulated by carbachol or dibutyryl-cAMP. T-593 depressed the maximal response of the histamine concentration-response curve with a dose-related displacement to the right, indicating that the nature of the H2-receptor antagonism of T-593 was insurmountable and included non-competitive inhibition. The inhibitory efficacy of T-593 was time-dependent and was retained after the cells were washed. The inhibitory potency of (-)-S-T-593, one of the enantiomers, on the [14C]aminopyrine accumulation stimulated by histamine was approximately twice that of racemic T-593 and it also behaved as an insurmountable H2-receptor antagonist. However, the potency of (+)-R-T-593 was markedly weak. These results suggest that T-593 has H2-receptor antagonism that is insurmountable and this agent slowly associates and dissociates with the receptor in isolated canine gastric mucosal cells and that the specific substance causing H2-receptor antagonism is (-)-S-T-593.     

Estimates of glycolysis, pyruvate (de)carboxylation, pentose phosphate pathway, and methyl succinate metabolism in incapacitated pancreatic islets

Pancreatic islets were cultured for 24 h in the presence of 1 mM glucose, which renders islets incapable of responding to glucose with insulin release. These islets were compared to islets maintained at 20 mM glucose for 24 h. Detritiation of [2-3H]glucose and [5-3H]glucose in 1 mM glucose islets was normal, suggesting that glucose transport and phosphorylation and all enzymes of glycolysis were not down-regulated in the incapacitated islets. 14CO2 formation from [U-14C]glucose and [6-14C]glucose was inhibited up to 80% and 14CO2 from methyl succinate was inhibited up to 60%, indicating that down-regulation at (a) mitochondrial site(s) might explain the incapacitated insulin release. 14CO2 formation from [3,4-14C]glucose (which becomes [1-14C]pyruvate) was decreased, indicating that the reaction catalyzed by pyruvate dehydrogenase was down-regulated. This decrease, however, was not as large as the decreases in 14CO2 formation from [U-14C]glucose, [2-14C]glucose (which becomes [2-14C]pyruvate), or [6-14C]glucose (which becomes [3-14C]pyruvate), indicating that other reactions were also down-regulated. 14CO2 formation from [1-14C]glucose was inhibited less than that from [6-14C]glucose in the incapacitated islets (34 vs 54%) and these rates indicated that flux of glucose through the pentose phosphate pathway was increased in the incapacitated islet, such that 29% (0.4 nmol of 1.4 glucose/100 islets/90 min) was metabolized via this pathway in the incapacitated islet but only 3.4% (0.1 of 2.9 nmol glucose/100 islets/90 min) was metabolized via the pentose pathway in the 20 mM glucose islets. With rates of 14CO2 evolved from glucose labeled at C2 and C6 and from methyl succinate labeled at C1 + C4 and C2 + C3 the 14CO2 ratio formula was used to calculate the ratios of carboxylated and decarboxylated pyruvate. Roughly equal amounts of pyruvate entered the citric acid cycle by each route in islets maintained for 24 h at 1, 5, or 20 mM glucose. The results indicate that decarboxylation and carboxylation of pyruvate were about equally suppressed in incapacitated islets and that direct inhibition of reactions of the cycle was unlikely. This is consistent with evidence which indicates that down-regulation of both pyruvate carboxylase and pyruvate dehydrogenase occurs in incapacitated islets, i.e., under long-term conditions that modify amounts of enzymes (MacDonald et al., 1991, J. Biol. Chem. 266, 22392-22397).(ABSTRACT TRUNCATED AT 400 WORDS)     

Roles of adrenoceptors in isolated canine parietal cells

Functional roles of adrenoceptors in parietal cells were pharmacologically investigated using isolated canine parietal cells. In the crude membranes obtained from preparations highly purified in parietal cells (> 95% of purity), the specific binding of [3H]dihydroalprenolol (DHA) was observed with a Kd value of 2.9 nM and Bmax of 234 fmol/mg protein, while the specific binding of [3H]prazosin and [3H]rauwolscine were not attained. Propranolol concentration-dependently reduced the specific binding of [3H]dihydroalprenolol with a Ki value of 2.6 nM. Isoproterenol concentration-dependently stimulated [14C]aminopyrine accumulation in preparations enriched in parietal cells (about 70% purity) with the maximum at 10 nM. Isoproterenol increased the content of cyclic AMP in preparations enriched in parietal cells (70%) with the maximum at 100 nM. The isoproterenol-induced stimulatory effect of [14C]aminopyrine accumulation in preparations enriched in parietal cells (70%) was completely abolished by 1 microM propranolol but not by 1 microM phentolamine. In the presence of 1 microM propranolol, 100 microM noradrenaline did not affect carbachol- and histamine-induced [14C]aminopyrine accumulation in preparations enriched in parietal cells (70%). The present study suggests that stimulation of beta-adrenoceptors located on canine parietal cells evokes acid production in a cyclic-AMP-dependent manner. Furthermore, a possibility arises that canine parietal cells are not the site of action of alpha-adrenoceptors in mediating inhibition of gastric acid secretion.     

Estimation of glucose turnover in rats in vivo with tritium labeled glucoses

Starved and starved-refed rats were injected intravenously with labelled glucose (a mixture of [2-3H]-, [3-3H]- and [U-14C]glucose with either [5-3H]- or [6-3H]glucose), and the decay of the specific activity of [14C]glucose followed. Glucose was degraded to obtain the 3H/14C ratios for 3 isotope combinations in the same sample. The apparent rates of replacements, apparent carbon recycling, and the body glucose mass were calculated for the different tracers. The 3H/14C ratio from [2-3H, -U-14C]glucose declined much faster than that of the other tracers. Apparent recycling as calculated in fasted rats was 28% for [2-3H, U-14C]- 18% for [5-3H,-U-14C]- 17% for [3-3H, U-14C]- and 14% for [6-3H,U-14C]glucoses. The values in fed rats showed a similar pattern. We estimate that in fasted rats 85 to 90% of the 3HOH liberated from injected [2-3H]glucose is formed by catabolism in the periphery and the rest by recycling in the liver between glucose and glucose 6-P. Detritiation of other labels by hepatic recycling accounts for a very small fraction of the total 3HOH yield.