Properties of 3H-cimetidine binding in rat brain membrane fractions

In an attempt to characterize the brain histamine H₂ receptor, experiments were undertaken to study the binding properties of (N-methyl-³H) -cimetidine, an H₂ receptor antagonist, in rat brain membranes. Using a centrifugation assay, ³H-cimetidine binding having a K_d of 0.40μM and a Bmax of 3.9 pmoles/mg protein was detected. Of fourteen anions and cations tested, one, Cu⁺⁺, dramatically increased specific ³H-cimetidine binding, the increase being due mainly to a change in Bmax. Studies of substrate specificity for ³H-cimetidine binding revealed that Cu⁺⁺, while not significantly affecting the potency of H₂ receptor agonists and antagonists, dramatically decreases the potency of H₁ receptor substances on the ³H-cimetidine binding site. In addition, both the relative and absolute potencies of various H₂ receptor agonistsv and antagonists in displacing the ligand in the presence of Cu⁺⁺ parallels their potencies in biological systems. These findings suggest that, under these conditions, ³H-cimetidine may be labelling a biologically relevant H₂ binding site in brain and that Cu⁺⁺ may regulate the substrate specificity for this site. The brain regional distribution and kinetic analysis of the binding suggest that it is not localized solely to the synaptic receptor for histamine, but may also be associated with histamine receptors at other neuronal, glial or vascular sites.     

Induction of translationally active rat liver glutathione S-transferase B messenger RNA by phenobarbital

Liver poly(A⁺)-RNA was isolated from untreated and phenobarbital-treated rats and translated in cell-free systems derived from wheat germ and rabbit reticulocyte lysates. The primary translation product of glutathione S-transferase B was comprised of two nonidentically sized subunits which comigrated on SDS-polyacrylamide gels with the purified glutathione S-transferase B subunits. The level of translatable glutathione S-transferase B mRNA in rat liver was elevated approximately 3 to 4-fold by phenobarbital administration. Our data suggest that chronic phenobarbital administration to rats increases the amount of cytosolic glutathione S-transferase B via an increase in the functional mRNA level encoding for the enzyme.     

Synthesis of liver cytochrome P-450b in a cell-free protein synthesizing system

Live ppolysomes isolated from rats that had been treated with phenobarbital (PB) are able to incorporate [³H]leucine into total protein $\text{in}\text{vitro}$ at a rate almost five times that of polysomes prepared from control animals. Specific immunoprecipitation of translational products has shown that polysomes from induced animals synthesize cytochrome P-450b at a rate almost seven times greater than polysomes from control animals. The increased protein and cytochrome P-450b synthesis can be detected as early as 6 h following phenobarbital administration and reaches a maximum at 12–18 h. The results suggest that PB administration effects an increase in mRNA for cytochrome P-450b.     

Discriminative effects of morphine administered intracerabrally in the rat

Rats were trained in a two-choice discrete trial avoidance paradigm to discriminate between saline and 3.0 mg/kg of morphine administered S.C. The microinjection of 0.3–3.0 μg of morphine into the lateral ventricle produced discriminative effects equivalent to those of the systemic training dose as measured by responding on the morphine-appropriate choice lever. Discriminative effects equivalent to those of the morphine training dose were not consistently produced by administration of morphine into the periaqueductal gray, lateral septum or dorsomedial thalamus in doses as high as 10 μg. However, the discriminative effects of systematically administered morphine were blocked by 10–30 μg of naloxone administered intracerebrally at all of the brain sites tested. Thus, the primary site at which morphine acts to produce discriminative effects in the rat is central, although the specific brain areas mediating these effects remain unidentified. The actions of naloxone could be the result of diffusion of the drug into the ventricular system or into the systemic circulation.     

Intestinal transport of zinc in the diabetic rat

Zinc has been implicated to play a role in the pathogenesis and management of diabetes. Since the intestinal transport of several minerals as calcium, magnesium and strontium was found to be altered in the diabetic rats, we postulated that intestinal zinc transport may be also altered in the diabetic rat. Therefore, using $\text{in}$$\text{vivo}$ single pass perfusion technique we determined lumen to mucosa flux, net absorption and the mucosa to lumen flux of zinc in the small and large intestinal segments of diabetic rats, diabetic rats treated with insulin and in control rats. Tissue distribution of transported ⁶⁵Zn into various organs and tissue concentrations of native zinc in the groups of rats studied were determined. Our results indicate that lumen to mucosa flux (μmole/h/g wet weight) was decreased in all intestinal segments of the diabetic rats compared to controls. However, the total capacity (mμmole/h/cm length) was similar. The specific activity and total capacity of net absorption of zinc was similar in all intestinal segments of the rats studied. The reverse mucosa to lumen flux was significantly decreased in all segments of diabetic rats compared to corresponding values in control rats. Tissue distribution of ⁶⁵Zn following the perfusion study showed increased retention of ⁶⁵Zn in the liver, kidney and femurs of the diabetic rats compared to controls. Serum and tissue concentration of native zinc in various organs were similar in all groups of rats studied. The mechanism(s) responsible for these findings are discussed.     

Stimulation of the hexose-monophosphate pentose pathway by delta 1-pyrroline-5-carboxylic acid in human fibroblasts.

L-pyrroline-5-carboxylic acid, an intermediate in the interconversions of glutamic acid, ornithine and proline, is a potent stimulator of the hexose-monophosphate pentose pathway in cultured human fibroblasts. These studies suggest that pyrroline-5-carboxylate reductase, which catalyzes the conversion of pyrroline-5-carboxylate to proline coupled with the oxidation of NADPH, provides the NADP for the observed activation of the hexose-monophosphate pentose pathway.     

NADH-cytochrome c reductase activity in cultured human lymphocytes: Similarity to the liver microsomal NADH-cytochrome b5 reductase and cytochrome b5 enzyme system

A NADH-cytochrome c reductase activity was increased upon mitogen stimulation of human lymphocytes. The activity was not inhibited by antimycin A or rotenone but was specifically inhibited by antibodies elicited against rat liver NADH-cytochrome b₅ reductase or cytochrome b₅. The activity was linear with cellular homogenates up to 5.2 × 10⁶ cells/ml and had abroad pH optimum of 7.7. The presence of 3-methylcholanthrene in mitogen stimulation media had no effect on the NADH-cytochrome c reductase activity but differentially induced the benzo(a)pyrene hydroxylase (AHH) activity. The reductase activity was present in nonstimulated cells and appears not to be significantly increased in activity per cell upon mitogen-stimulation of the peripheral lymphocyte.     

Stimulation by polydeoxyribonucleotide of histone phosphorylation by guanosine 3':5'-monophosphate-dependent protein kinase.

[³H]Dihydroalprenolol bound to a single population of high affinity sites in rat myocardial membranes when the concentration of the radioligand was below 5 nM. These sites displayed characteristics which would be expected of binding to the β-receptor. Kinetic- and Scatchard-derived dissociation constants were 0.6 and 2.0 nM, respectively. Binding was to a limited number of sites, 60 fmols/mg protein. Scatchard analysis using radioligand concentrations in excess of 5 nM resulted in concave upward plots suggestive of more than one population of binding sites. The lower affinity sites (labeled at high radioligand concentration) were non-stereospecific in nature and became a progressively larger fraction of “specific binding” as the concentration of dihydroalprenolol was increased above 5 nM.     

Interactions of cadmium and selenium in rat plasma in vivo and in vitro

⁷⁵Se and ¹⁰⁹Cd tracers were used to study the binding of Se and Cd to plasma proteins at various SeO₃^2? doses and times up to 24 h after the simultaneous subcutaneous administration of SeO₃^2? and CdCl₂ to adult male rats. The simultaneous injection of CdCl₂ and SeO₃^2? markedly increased both Se and Cd plasma levels over that in control animals. Gel permeation chromatography of plasma indicated that at all times up to 24 h Cd and Se were bound in an atomic ratio of approx. 1 : 1 in 330 000 and 130 000 dalton fractions. From 4 to 24 h, Cd and Se appeared in the 420 000 dalton fraction, also with an atomic ratio of approx. 1 : 1. The 330 000 dalton molecules appeared to have a maximal binding capacity for the Cd-Se complex at a concentration of approx. 30 μmol/ml of plasma, while the 130 000 and 420 000 dalton molecules show a higher binding capacity. Studies in vitro revealed that SeO₃^2? does not interact directly with Cd and plasma proteins. It is metabolized by erythrocytes to a form that interacts in an atomic ratio of 1 : 1 with Cd to form a protein-bound complex of 130 000 daltons.     

Multiple forms of cyclic nucleotide phosphodiesterases in normal and goitrous rat thyroid

The stoichiometry of H⁺ ejection coupled to electron flow from succinate to ferricyanide in the electron transport chain of mitochondria from Ehrlich ascites tumor and AS30-D hepatoma cells was determined. Values close to 4.0 for the $\text{H}{}^{\mathrm{+}}\text{2e}{}^{\mathrm{?}}$ ejection ratio were found in both cell lines, with either Ca²⁺ or K⁺ (+ valinomycin) as charge-compensating permeant cation. The 4 H⁺ ejected were compensated by outward movement of two negative charges to reduce 2 Fe(CN)6^3? to 2 Fe(CN)6^4?, and the uptake of two positive charges in the form of the permeant cation. Experiments on (a) omission of rotenone (b) the effect of antimycin A and (c) depletion of endogenous NAD(P)-linked substrates showed that no significant endogenous electron flow or H⁺ ejection occurred, thus eliminating possible overestimation of the H⁺/2e^? ratio from endogenous substrates. These data on mitochondria from two tumor cell lines are fully consistent with earlier measurements of the H⁺/O stoichiometry for succinate and NADH oxidation in tumor mitochondria and with the $\text{H}{}^{\mathrm{+}}\text{2e}{}^{\mathrm{?}}$ stoichiometry for site 2 in normal rat liver mitochondria.     

Stimulation of myometrial and decidual prostaglandin production by amniotic fluid from term,but not midtrimester pregnancies

The effect of amniotic fluid obtained from second trimester (16–20 wks) and term pregnancies (38–41 wks) on the production of PGE and F by human amnion, decidua and myometrium at term was determined using tissue slices incubated in vitro. Midpregnancy amniotic fluid neither inhibited nor stimulated the prostanoid production by any of the tissues. In contrast, term amniotic fluid obtained before as well as after the onset of labor markedly increased the production of both PGE and PGF in decidua and myometrium from levels in Krebs solution. The prostanoid production (PGE + PGF) in amnoin was not significantly increased but the proportion of PGF was raised during incubations in term amniotic fluid. In decidua and myometrium the increase in PGE and PGF production in term amniotic fluid was approximately 200 and 400 percent respectively, from control values in Krebs solution. We propose that the stimulatory activity in term amniotic fluid in responsible for the accelerated synthesis of prostaglandins after of membranes, which is reflected in raised PGF metabolite levels in maternal circulation. It may also be the reason for the rise in amniotic fluid prostaglandin levels around the 35th week of gestation, and perhaps for the onset of labor.     

Diurnal variations in inducibility of rat liver tyrosine aminotransferase activity.

The activity of tyrosine aminotransferase (TAT) was measured in the livers of rats which were entrained to eat for the first 2 hours of a daily 12 hour dark period (‘2+22’ schedule) and were treated with the synthetic glucocorticoid dexamethasone and with glucagon at several times of day. TAT activity in untreated animals varies diurnally with a maximum 4 to 6 hours after the beginning of feeding. In both fed and fasted rats there was a small diurnal variation in inducibility by dexamethasone: in fed rats induction was greatest near the beginning of the dark period, shortly after feeding; in fasted rats induction increased towards the end of the dark period. Glucagon induction showed a marked diurnal variation in fed rats with a decrease coincident with the decline in control TAT activity after its food-induced peak. This variation did not appear to be depemdent on food intake, however, since the decline in inducibility occurred in fasted rats at the same time as in fed rats. Co-treatment with dexamethasone did not affect the decrease in glucagon inducibility. The diurnal variation in TAT induction may reflect a diurnal rhythm in the components of the enzyme synthesizing system (e.g. in the availability of mRNA or in enzyme degradation).     

The in vivo transformation of phospholipid vesicles to a particle resembling HDL in the rat.

The tumor-specific transplantation antigen (TSTA) in crude three molar potassium chloride (3M KCl) extracts of a chemically-induced, murine fibrosarcoma was purified by ammonium sulfate salt fraction at 20% saturation (S20S) and by polyacrylamide gel electrophoresis (PAGE). Mice, which had been pretreated with the S20S precipitate, displayed retarded outgrowth of a 100-fold supralethal dose of the corresponding, but not of a non-cross-reactive syngeneic tumor. Analysis of PAGE gels by Coomassie Blue staining revealed at least 30 bands in the crude 3M KCl extract, and only two components (Rf 0.34 and 0.43) in the ammonium sulfate fraction. That these two components bore TSTA activity was demonstrated by the observation that the immunoprotective activity of crude 3M KCl extracts was localized to the Rf 0.25–0.50 region. The two components present in the S20S fraction had isoelectric points of 5.05 and 6.9, and estimated molecular weights of 40,000 and 75,000, thus demonstrating the soluble nature of the active principle. These findings offer the prospect of a chemical dissection of the polymorphic TSTA surface markers on MCA-induced murine tumors.     

The correlation between plasminogen activator-stimulated DNA synthesis and cell morphology in 3T3 cells

The internalization of plasma membrane components labelled with ConA and peroxidase was investigated in monolayer cultures of rat liver cells. After the labelling procedure, the cells were reincubated with PBS free of both ConA and peroxidase for different time periods between 5 min and 3 h at 37 °C. Ligand-induced redistribution of ConA-binding sites finally resulted in a cap with uropod formation after 2–3 h of reincubation. Simultaneously with redistribution, the cell surface label disappeared through internalization, and a membrane recycling into the Golgi apparatus could be observed. Besides the lamellar Golgi apparatus which exhibited a labelling of the cisternae as a consequence of the membrane recycling, the hypertrophied unlabelled Golgi apparatus could be detected in the same cell. Furthermore, many vesicles formed by the hypertrophied Golgi apparatus were found between them and the plasma membrane and in close proximity to the plasma membrane. Fusion of the vesicles with the plasma membrane could be observed. These morphological findings indicate the possibility that the membrane internalization and the membrane recycling simultaneously effect an enhancement of membrane biogenesis and exocytosis, thus compensating for the membrane removal by internalization.     

Effect of acyclic polyisoprenoids on the biosynthesis of mannose-labeled glycolipids in rat liver microsomes

The effect of the acyclic polyisoprenoid (C₂₀); geranylgeranyl-acetone (GGA) and its derivatives on mannolipid formation from GDP-mannose in rat liver microsomes was studied. Remarkable increases in mannolipid biosynthesis (Rf 0.24 and/or Rf 0.50) were observed by $\text{in vitro}$ additions of GGA, its hydroxylated compound (GGA-OH) and phosphorylated material (GGA-OP). These results strongly suggest that GGA-OP is involved in mannolipid formation (Rf 0.24) as an acceptor, in analogy to retinylphosphate. On the other hand, the mechanism for increased formation of an endogenous mannolipid (Rf 0.50) by GGA and GGA-OH is attributable to the enhancement of mannosyltransferase activity.     

Effect of urethan on polyamine and DNA synthesis in the regenerating rat liver

When a single dose of urethan was injected into the peritoneal cavity of rats immediately after partial hepatectomy, DNA synthesis was delayed by 12 h. The induction of ornithine decarboxylase which was induced biphasically following partial hepatectomy was also reduced and delayed by 14–15 h by the administration of urethan. S-Adenosylmethionine decarboxylase activity in urethan-treated rat liver at 20 h and 29 h after operation was significantly lower than that of untreated animals. This enzyme activity was shown to increase thereafter, reaching a higher level than in untreated rats at 37–42 h. Hepatic spermidine content changed biphasically in a manner similar to DNA synthesis. These results suggest that the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase may correlate with DNA synthesis and that an increase of spermidine concentration is necessary to DNA synthesis.     

A serotonin sensitive adenylate cyclase in mature rat brain synaptic membranes

Results from this study have indicated serotonin-sensitive adenylate cyclase activity in adult rat brain. The enzyme is localized in the synaptosomal plasma membrane and apparently has multiple activation sites for serotonin with specific activity maxima occuring at serotonin concentrations of 5 × 10^?10, 5 × 10^?9, 1 × 10^?8, and 5 × 10^?8 moles/liter. The production of cyclic AMP at these sites appears to be unaffected by 1 × 10^?5M fluphenazine, while 1 × 10^?5M tryptamine, methysergide, and ergonovine decreased the stimulatory effect of 1 × 10^?8M 5-HT by 30 percent, 80 percent, and 57 percent respectively.     

Vitamin A-dependent fucose-glycopeptide from rat tracheal epithelium

Tracheas from normal and vitamin A-deficient rats were incubated in the presence of l-[³H]fucose and d-[¹⁴C]glucosamine to label epithelial and mesenchymal glycoproteins. The epithelium and the cartilage were separated by incubation with testicular hyraluronidase and processed separately for the preparation of glycopeptides. One major epithelial glycopeptide was doubly labeled. In vitamin A deficiency the amount of l-[³H]fucose was reduced to 33% that of the normal. The tracheal fucose-glycopeptide, in molar ratios, contained fucose 1.0, mannonse 1.0, galactose 0.4, hexasamine 4.7, and sialic acid 3.5. Sulfate was absent.