Binding of vasoactive intestinal peptide and its stimulation of adenylate cyclase through two classes of receptors in rat liver membranes effects of 12 secretin analogues and 2 secretin fragments

1. Vasoactive intestinal peptide (VIP) receptors were identified in crude rat hepatic membranes by ¹²⁵I-labelled VIP binding and by the ability of VIP to stimulate adenylate cyclase activity. The specificity of these receptors was evaluated by the capacity of secretin, synthetic secretin analogues, and secretin fragments to inhibit ¹²⁵I-labelled VIP binding and to stimulate adenylate cyclase. 2. The results were compatible with the existence of two classes of VIP binding sites that could be distinguised according to their affinity for VIP and their specificity. High-affinity sites were more specific for VIP as secretin was 175 times less potent than VIP for recognition of these sites while being only 33 times less potent than VIP for recognition of low-affinity sites. 3. Secretin analogues, monosubstituted in position 2, 3, 4, or 6 were less potent than secretin for adenylate cyclase stimulation as well as for the recognition of the two classes of receptors. [Val⁵]Secretin was more potent than secretin and appeared definitely more VIP-like than secretin; [Ala⁴, Val⁵]secretin were equipotent to secretin. 4. The fragment secretin (7–27) was unable to recognize VIP receptors and to stimulate adenylate cyclase. The substituted fragment [Gln[⁹,Asn¹⁵]secretin (5–27) recognized these receptors with weak potency but could not activate the enzyme.     

Stimulation by 3-mercaptopicolinate of net glusose uptake by perfused livers from diabetic rats

Glucose uptake/production was studied as a function of varied glucose loadsin isolated perfused livers from glucagon-treated alloxan-diabetic rats. Uptake of D-[U-¹⁴C]glucose was seen at all levels studied - 9.5–71 mM. In studies with unlabelled D-glucose carried out in the absence of 3-mercaptopicolinate, livers of diabetic rats showed a net production of glucose with perfusate glucose levels less than 22 mM. Above this level, these livers exhibited a time- and concentration-dependent net uptake of glucose for the period of 20–30 min. When 4 mM 3-mercaptopicolinate, which inhibited gluconeogenesis from endogenous substrates, was included in perfusates, a continuous net uptake of unlabelled glucose was observed at all levels above 4 mM. This lowering of the null-point, cross-over glucose concentration was shown to relate mechanistically to the observed reduction in steady hepatic glucose 6-phosphate level produced by mercaptopicolinate. The need for supplemental mechanisms of glucose utilization by high K_ww hepatic enzyme(s) operative in the virtual absence of insulin-dependent glucokinase also is indicated by these observations and by kinetic analysis.     

Phosphoenolpyruvate synthesis by fetal guinea-pig liver mitochondria

Liver mitochondria isolated from fetal and newborn guinea pigs synthesized phosphoenolpyruvate at 4–6 nmol/min per mg protein with 2 mM malate, succinate, and α-ketoglutarate as substrates. These rates were 90–110% of that by adult liver mitochondria and were not substantially altered in the second half of gestation or within 24 h after birth. Both palmitoyl- and octanoylcarnitine were inhibitory to phosphoenolpyruvate synthesis in adult and fetal preparations, but free octanoate was inhibitory only in adult liver mitochondria.     

The involvement of parenchymal,Kupffer and endothelial liver cells in the hepatic uptake of intravenously injected liposomes effects of lanthanum and gadolinium salts

¹²⁵I-labeled albumin or poly(vinyl pyrrolidone) encapsulated in intermediate size multilamellar or unilamellar liposomes with 30–40% of cholesterol were injected intravenously into rats. In other experiments liposomes containing phosphatidyl[Me-¹⁴C]choline were injected. 1 h after injection parenchymal or non-parenchymal cells were isolated. Non-parenchymal cells were separated by elutriation centrifugation into a Kupffer cell fraction and an endothelial cell fraction. From the measurements of radioactivities in the various cell fractions it was concluded that the liposomes are almost exclusively taken up by the Kupffer cells. Endothelial cells did not contribute at all and hepatocytes only to a very low extent to total hepatic uptake of the ¹²⁵I-labels. Of the ¹⁴C-label, which orginates from the phosphatidylcholine moiety of the liposomes, much larger proportions were recovered in the hepatocytes. A time-dependence study suggested that besides the involvement of phosphatidylcholine exchange between liposomes and high density lipoprotein, a process of intercellular transfer of lipid label from Kupffer cells to the hepatocytes may be involved in this phenomenon. Lanthanum or gadolinium salts, which effectively block Kupffer cell activity, failed to accomplish an increase in the fraction of liposomal material recovered in the parenchymal cells. This is compatible with the notion that liposomes of the type used in these experiments have no, or at most very limited, access to the liver parenchyma following their intravenous administration to rats.     

Modulation by somatostatin of nerve-mediated activation of glycogenolysis in the perfused rat liver

Perivascular nerve stimulation of rat livers perfused in situ with erythrocyte-free Krebs-Henseleit buffer at constant pressure in a non-recirculating system resulted in an increase of glucose and lactate production and in a decrease of portal flow. Infusion of somatostatin in different concentrations (2 × 10⁻⁷, 10⁻⁸, 10⁻⁹ mol·l⁻¹) reduced the nerve-mediated activation of glucose release maximally to 66%. There was only a slight effect on the lactate output, the nerve-mediated reduction of portal flow was unaltered. In controls, somatostatin alone had no effect on the metabolic and hemodynamic parameters. In order to differentiate between a presynaptic and postsynaptic mechanism, the noradrenaline overflow was calculated. The unaltered release of the neurotransmitter in the presence or absence of somatostatin excluded a presynaptic mechanism. To mimic the nerve effects on the carbohydrate metabolism and on the hemodynamics, noradrenaline (2 × 10⁻⁷ mol·l⁻¹) was infused instead of the nerve stimulation over a period of 5 min. Somatostatin did not change the endocrine effects of the catecholamine under these conditions. The nerve-dependent effect of somatostatin suggests that other neurotransmitters (e.g. VIP) or mediators (e.g. prostanoids) may be influenced by somatostatin.     

Uptake of asialoglycophorin-liposomes by the perfused rat liver

Asialoglycophorin-containing liposomes and their contents (¹²⁵I-labeled bovine serum albumin) were taken up by a perfused rat liver with subsequent digestion of their protein components. The uptake of these liposomes required Ca²⁺ as well as desialylation. The process was inhibited partially by asialofetuin and completely by further addition of asialoglycophorin to the perfusate.     

Isolation of a rat liver plasma membrane fraction of probable canalicular origin Preparative technique,enzymatic profile,composition, and solute transport

A technique currently used for isolation of brush border membranes from renal and intestinal epithelium that involves vigorous tissue homogenization and sedimentation of non-luminal membranes in the presence of Mg²⁺ has been adapted to rat liver. Liver plasma membranes so prepared consisted almost exclusively of vesicles by electron microscopy, showed some contamination with endoplasmic reticulum and minimal contamination with mitochondria or Golgi by marker enzymes, were highly enriched in alkaline phosphatase, Mg²⁺-ATPase, and 5′-nucleotidase activity compared with homogenate, and showed little enrichment in (Na⁺,K⁺)-ATPase. Comparison of this enzymatic profile with cytochemical studies localizing (Na⁺,K⁺)-ATPase and alkaline phosphatase to the sinusoidal/lateral and canalicular membranes, respectively, suggested that these membranes were predominantly of canalicular origin. They had a lower ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ specific activity, lower lipid content, and higher cholesterol to phospholipid molar ratio than a conventional plasma membrane preparation believed to be enriched in canaliculi. Moreover, it was possible to measure movement of d-[³H]glucose into an osmotically sensitive space bounded by these membrane vesicles.     

Purification of protein synthesis initiation factor eIF-3 from rat liver microsomes by affinity chromatography on rRNA-cellulose

An efficient four-step procedure is described for preparing highly purified polypeptide chain initiation factor eIF-3 from rat liver microsomal saltwash. The method involves fractionation with ammonium sulfate between 25–40% saturation (0°C) followed by affinity chromatography on rRNA-cellulose, DEAE-cellulose chromatography and sucrose density gradient centrifugation. eIF-3 is eluted from the affinity column at a KCl concentration of 0.18 M. The purification is 10-times and the recovery of activity better than 85%. In the sucrose gradients, eIF-3 sediments as a 15 S particle indicating a total mass of 650 000 Da. The purified eIF-3 is highly active in stimulating globin synthesis in a fractionated translation system. Factor eIF-3 contains eight subunits with molecular weights ranging from 40 000 to 110 000. Seven of the subunits are present in one copy per eIF-3, whereas the factor contains two copies of one subunit. The isoelectric points of the factor subunits range from 5.5 to 7.3 with most of the polypeptides being acidic.     

High-affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin in cell nuclei from rat liver

The intranuclear binding of radioactive 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat liver has been studied both in vivo and in vitro. Following the intravenous administration of [1,6-³H]TCDD, a maximum uptake by cell nuclei could be observed at 2 h after injection with a concurrent decrease in the cytosolic uptake. Using linear sucrose density gradient centrifugation, dextran-coated charcoal adsorption assay, DEAE-Sepharose ion-exchange chromatography, competition, enzymatic and saturation studies, a high-affinity binding protein for TCDD in liver cell nuclei could be demonstrated both in vivo and after an exchange in vitro of intravenously administered unlabelled 2,3,7,8- tetrachlorodibenzofuran (TCDBF) for [³H]TCDD. Sucrose density gradient analysis showed a size of 4–5 S for both the cytosolic and nuclear TCDD binding entity. The specific binding of [³H]TCDD to nuclear components was heat labile and saturable and had an equilibrium dissociation constant of 1.05 nM. Based on a differential susceptibility to specific hydrolases, i.e. DNAase, RNAase, trypsin and pronase, the binding entity appears to be a 4–5 S salt-extractable protein.     

Uptake of oxidized folates by rat liver mitochondria

Folate, dihydrofolate, and methotrexate are rapidly taken up by rat liver mitochondria. The apparent maximal matrix folate concentration is about 2.5-fold that of the suspending medium, whereas dihydrofolate and methotrexate equilibrate across the inner membrane. Fully reduced folates, including tetrahydrofolate, 5-methyltetrahydrofolate, and 5,10-methylenetetrahydrofolate penetrate only the intermembrane space. Addition of dihydrofolate or methotrexate effects a rapid release of pre-loaded folate, and external methotrexate promotes the release of pre-loaded dihydrofolate. The extent of dihydrofolate uptake is enhanced by addition of folate. These results suggest that oxidized folates are transported to the matrix by a carrier-mediated mechanism.     

Determination of Ca2+- and phospholipid-dependent protein kinase in rat liver membranes

A method has been developed to measure the Ca²⁺- and phospholipid-dependent protein kinase in membrane fractions. The method is based on the fact that this enzyme is resistant to comparatively high concentrations of octyglycoside. Rat liver membranes were treated with octylglycoside and the phosphate incorporation from |-³²P]ATP was measured in the presence of histone H1. The enzyme activity was determined as the difference between the incorporation obtained after addition of Ca²⁺ and phosphatidylserine and the incorporation obtained without these additions but with EGTA. The endogenous incorporation of phosphate to membrane components was constant under these incubation conditions. The conditions for determination of the membrane-bound enzyme were optimized. Two thirds of the total enzymic activity was attached to membranes in rat liver cells. A highly purified plasma membrane preparation had the highest specific activity, while most of the bound enzyme was found in microsomes, an only traces were found in mitochondria.     

Changes in the sequence content of albumin mRNA and in its translational activity in the rat liver with age

To investigate the regulation of age-related changes in albumin synthesis in the rat liver, total postnuclear RNA and polyribosomes, both membrane-bound and free, were prepared from livers of rats of different ages. By the use of a specific complementary DNA probe, the albumin mRNA sequence content was quantitated in these RNA fractions. These studies showed a specific increase in albumin mRNA sequence content in total postnuclear RNA and membrane-bound polyribosomes at between 12 and 24 months of age. Between 24 and 36 months of age, the increase in the amount of albumin mRNA in these two fractions was due only to an increase in liver weight. The increase in albumin mRNA sequence content was not found in the poly(A)⁺ fraction but in the RNA extracted from the void of oligo(dT)-cellulose column chromatography. The isolated polyribosomes were translated in a cell-free system to assess age-related changes in total protein and albumin synthesis due to translational control. No changes with age were found in the translational capacity of membrane-bound and free polyribosomes per RNA unit. Immunoprecipitation of the synthesized albumin in the translation products revealed that albumin synthesis in the cell-free system is not increased proportionally with the elevated albumin mRNA level between 12 and 24 months of age. This indicates that albumin mRNAs present in the livers of old rats are biologically less active than those found in younger animals.     

A steady state and differential polarised phase fluorimetric study of the liver microsomal and mitochondrial membranes of thermally acclimated green sunfish (Lepomis cyanellus)

The liver mitochondrial and microsomal membranes of green sunfish and rat were examined by steady state polarisation and differential polarised phase fluorimetry to determine the effects of seasonal adaptation of membrane dynamic structure to temperature. Steady state polarisation studies indicated that the liver mitochondria of green sunfish acclimated to different temperatures showed a greater partial compensation of membrane fluidity for the altered acclimation temperature than did liver microsomal membranes. The fatty acid composition of both membrane preparations generally became more unsaturated at lower acclimation temperatures, though the differences between 5°C and 25°C acclimated fish were more pronounced in the mitochondrial fraction than in the microsomal fraction.Differential polarised phase fluorimetric studies indicated that the rotations of diphenylhexatriene in mitochondrial and microsomal membranes were highly hindered, though the hindrance offered by membranes of 25°C acclimated green sunfish was far greater than that offered by the membranes of 5°C acclimated fish, thus supporting the concept of homeoviscous adaptation. The absolute rotational rate was not consistently affected by acclimation treatment.     

Effect of anions on folate binding by isolated brush border membranes from rat kidney

The characteristics of folate binding by brush border membranes from rat kidney homogenates were investigated. At pH 7.4, binding of [3′, 5′, 9-³H]-pteroylglutamic acid to membranes containing endogenous folate is inhibited by anions, with chloride being most effective followed by bromide, thiocyanate, iodide, phosphate and sulfate. A maximum inhibition of 70–75% is attained at a concentration of 0.1 M chloride and an incubation time of 30 min. The inhibition diminishes with increased incubation time and at 24 h is negligible. The binding of [3′,5′,9-³H]pteroylglutamic acid to brush border membranes stripped of endogenous folate by acid treatment is not inhibited by anions. Anion sensitivity can be restored to these treated membranes by reconstitution with membrane-derived folate, particularly 5-methyltetrahydropteroylglutamic acid, or by preincubation with synthetic 5-methyltetrahydropteroylglutamic acid. Inhibition of [3′,5′,9-³H]pteroylglutamic acid binding by anions in membranes with endogenous folate is best explained by an anion-induced stabilization of endogenous folate-binding protein complex resulting in a decreased rate of exchange with exogenous [3′,5′,9-³H]pteroylglutamic acid.     

An improved purification and further characterization of the messenger ribonucleic acid for the fatty acid synthetase from rat liver

High purity fatty acid synthetase mRNA has been prepared from rat liver. The translational purity of the mRNA preparation was at least 27% as judged by the percentage of the radioactivity incorporated into acid-insoluble material that was precipitated by anti-fatty acid synthetase antibody. The specific activity of the mRNA was 220-times greater than that reported previously from this laboratory [1]. The large increase in the specific activity was achieved by the repeated use of high resolution linear-log sucrose density gradient centrifugation and the removal of 28 S rRNA by Sepharose 4B chromatography, as well as by the optimization of the K⁺ concentration (160 mM) in the reticulocyte lysate translation system. The mRNA preparation showed a single major band on agarose gel electrophoresis under denaturing conditions, and the translational activity of the fatty acid synthetase mRNA on the gel was found to coincide with this band. The molecular weight of the fatty acid synthetase mRNA is 2.5·10⁶ Da. The mRNA directed the synthesis of fatty acid synthetase with a molecular weight indistinguishable from that of the authentic enzyme subunit (M_r = 240 000). The copurification of the translation product and authentic enzyme revealed that the fatty acid synthetase polypeptides synthesized in the reticulocyte lysate system are assembled in vitro into dimers, the native form of the enzyme.     

Sequestration of adenosine in crude extract from mouse liver and other tissues

Adenosine (1 μM) was incubated in the presence of dialyzed crude tissue extract from mouse liver and its degradation determined. At high concentration of tissue extract, a fraction of adenosine was not metabolized. This phenomenon, termed sequestration of adenosine, was shown to be affected in the same way by the same factors (pH, salt, reducing agent and adenine) as those affecting the protection of adenosine against deamination in the presence of the purified cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase from mouse liver (Sæbø, J. and Ueland, P.M. (1979) Biochim. Biophys. Acta 587, 333–340). These data point to a role of this protein in the sequestration of adenosine in crude extract.The sequestration potency in crude extract could be determined by diluting the extract in the presence of a constant amount of adenosine deaminase added to the tissue extract. Under these conditions there was linearity of adenosine not available for degradation versus the concentration of tissue extract, and a total recovery of the sequestration potency of purified binding protein added to the crude extract was observed.The tissue level of the cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase in mouse liver was determined by two independent procedures based on the sequestration of adenosine and the hydrolysis of S-adenosylhomocysteine, respectively. The intracellular concentration was calculated to be 10 μM.The sequestration of adenosine in crude extract from mouse, rat, rabbit and bovine tissues was determined and showed requirements similar to those of the sequestration in mouse liver extract.The ability to sequester adenosine was high in liver and decreased in the following order: liver, kidney, adrenal cortex, brain, uterus, cardiac and skeletal muscle.     

Intracellular transport and degradation of 125I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

Triiodothyronine nuclear receptor: Role of histones and DNA in hormone binding

The triiodothyronine (T₃) nuclear receptor was previously shown to lose rapidly its high affinity hormone-binding property after a partial purification from the nuclear extract. It was then found that histones + DNA added to the incubation medium with labeled T₃ could restore, at least in part, the high affinity T₃ binding. We now demonstrate that DNA alone increases the high affinity T₃ binding site concentration moderately, and only at low ionic strength where it can bind to the receptor. Total histones and all histone fractions studied (total core histones, F2a, H2B, H3, H4, H1) specifically increase, at low concentrations, the level of T₃ binding; but higher concentrations of some individualized histones, particularly arginine-rich histones, have an inhibitory effect. DNA, or several other polynucleotides, in the presence of histones increase the stimulating histone effect and reverse the inhibitory effect into a true activation. Histones increase the number of T₃ binding sites but decrease the affinity for T₃; addition of DNA restores the high affinity for T₃ and stabilizes the T₃-receptor complexes. Thus, some of the histone molecules could play a role in the maintenance of the T₃ binding site, but multiple interactions between histones or with DNA seem necessary to impair the negative effect exerted by other parts of the histone molecules. Whether these positive and negative effects of histones on the T₃ binding site are of biological relevance in the regulation of T₃ binding to its receptor remains to be determined.     

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells

Receptor sites for insulin on GH₃ cells were characterized. Uptake of ¹²⁵I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37°C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with ¹²⁵I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for ¹²⁵I-labeled insulin binding sites. ¹²⁵I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. ¹²⁵I-labeled insulin was degraded at both 4 and 37°C by GH₃ cells, but not by medium conditioned by these cells. After a 5 min incubation at 37°C, products of ¹²⁵I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of ¹²⁵I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of ¹²⁵I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of ¹²⁵I-labeled insulin, as well as intact hormone, were recovered from GH₃ cells. After 30 min incubation at 37°C, 80% of the cell-bound radioactivity was not extractable from GH₃ cells with acetic acid.     

Topography of somatostatin cells in the stomach of the rat: Possible functional significance

Summary Somatostatin cells in the stomach of the rat have a characteristic shape and distribution. In the antral mucosa they occur together with gastrin cells and enterochromaffin cells at the base of the glands. In the oxyntic mucosa they are scattered along the entire glands with some predominance in the zone of parietal cells. Throughout the gastric mucosa the somatostatin cells possess long and slender processes that emerge from the base of the cell and end in clublike swellings. Such processes appear to contact a certain proportion of neighbouring gastrin cells in the antral mucosa and parietal cells in the oxyntic mucosa.Exogenous somatostatin given by intravenous infusion to conscious rats counteracted the release of gastrin stimulated by feeding, elevated antral pH or vagal excitation. Gastrin causes parietal cells to secrete HCl and endocrine cells in the oxyntic mucosa to mobilise and synthesise histamine. Somatostatin is known to block the response of the parietal cells to gastrin. In contrast, somatostatin did not block the response of the histamine-storing endocrine cells to gastrin, perhaps because these endocrine cells lack receptors to somatostatin. Conceivably, somatostatin in the gastric mucosa has a paracrine mode of action. The observations of the present study suggest that somatostatin may affect some, but not all of the various cell types in the stomach. Under physiological conditions this selectivity may be achieved in the following ways: 1) Communication may be based on direct cell-to-cell contact. 2) Only certain cell types are supplied with somatostatin receptors.