Changes in [3H]galactose uptake in human colonic mucosa with carcinoma: An ultrastructural study

Summary As part of our investigation on glycoprotein synthesis in pre-malignant colonic epithelium, changes in the uptake of [³H]galactose were studied at the ultrastructural level. Normal control mucosa from rectal biopsies of patients with no known gastro-intestinal disease and mucosa adjacent to carcinoma (transitional mucosa) from specimens resected for colo-rectal cancer were compared. These tissues were incubated in TC 199 medium containing [³H]galactose for various intervals of time and for pulse labelling. Silver grain distribution was statistically analysed. The results showed a reduction in the incorporation of the galactose by transitional mucosa. The uptake by this mucosa was less uniform than normal and showed considerable peaking in the endoplasmic reticulum of the goblet cells and in the Golgi of the absorptive cells, suggesting a blockage or alteration in glycoprotein synthesis. The differences were most marked in the middle crypt (the region of differentiation) and in the upper crypt (the region of maturation).     

Intracellular disposition of [3H]-cocaine, [3H]-norcocaine, [3H]-benzoylecgonine and [3H]-benzoylnorecgonine in the brain of rats

[³H]-cocaine, [³H]-norcocaine, [³H]-benzoylecgonine and [³H]-benzoylnorecgonine were administered i.c. in equi-potent pharmacologic doses and the intracellular disposition and metabolism of each drug determined. Norcocaine and cocaine rapidly entered and egressed from the brain so that 4.8–6.1% of the radioactivity present in brain at one minute was observed at 30 minutes. The highest levels of subcellular radioactivity were generally found in the microsomal plus supernatant, followed by the nuclear and shocked mitochondrial fractions. No apparent localization of the radioactivity occured in synaptic membranes. The brain/plasma (B/P) ratio curves for cocaine and norcocaine were similar; however, the norcocaine values were considerably higher at each time interval. Benzoylecgonine and benzoylnorecgonine had higher comparative B/P ratios than cocaine or norcocaine and persisted in brain for a longer period of time so that 0.6–2.1% of the radioactivity present in brain at 1 hour was detected at 24 hours. Cocaine and norcocaine were extensively metabolized to the benzoylmetabolites. Benzoylecgonine was metabolized to benzoylnorecgonine and benzoylnorecgonine was unmetabolized. The brain disposition data and B/P ratios agreed quite well with the overall pharmacologic action of cocaine and its metabolites.     

A comparison of [3H]galactose and [3H]fucose uptake with the morphological and histochemical changes observed in mucous secretion in chemically induced rat colonic carcinoma

Summary The alterations in carbohydrate metabolism which occur in the distal colon of rats during carcinogenesis induced by dimethylhydrazine were investigated using [³H]galactose and [³H]fucose as glycoprotein precursors.A statistically significant decrease in [³H]galactose uptake was observed in dysplastic epithelia. These findings are consistent with the alterations in mucin composition with predominance of sialomucins shown in these areas by histochemical methods. Furthermore, changes in the gradient of [³H]galactose incorporation along the crypt epithelium were also found in the histological and histochemically non-involved colonic mucosa of dimethylhydrazine-treated rats, as compared with controls.No significant variations were seen in [³H]fucose incorporation.These results correlate well with our previous histochemical observations and are further evidence of the profound alterations in glycoprotein synthesis affecting the whole colonic mucosa during carcinogenesis.     

Dopamine receptors in the rat brain: quantitative autoradiographic localization using [3H]sulpiride

In vitro labeling of tissue sections with [³H]sulpiride has been utilized in the present study to autoradiographically localize D₂-dopamine receptors in the rat brain. Preliminary biochemical studies, using slide-mounted tissue sections, were performed to define the optimal labeling conditions for this binding. Autoradiograms were generated by apposition of the labeled tissue sections to tritium-sensitive film. Specific binding sites for [³H]sulpiride were localized to the caudate-putamen, nucleus accumbens, olfactory tubercle, glomerular layer of the olfactory bulb, pituitary, laminae I and III of the entorhinal cortex, substantia nigra, lateral mammillary nucleus and the stratum-lacunosum moleculare of the hippocampus. The high selectivity of [³H]sulpiride for the D₂-dopamine receptor indicates that it is a valuable tool for the autoradiographic localization and quantitation of neuroleptic receptors.     

Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [³H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [³H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.     

Preferential release of newly synthesized [3H]GABA from striatal slices to preloaded [3H]GABA

The release of [³H]GABA which is newly synthesized from [³H]l-glutamic acid (GLU) has been examined using striatal slices obtained from the rat brain. It was found that 8–10% of [³H]GLU transported was converted to [³H]GABA during the incubation of striatal slices in the presence of nipecotic acid (5 × 10^?5 M). Nipecotic acid was added to the medium in order to prevent possible reuptake of [³H]GABA released during its synthesis, and it was found to have no significant effect on the formation of [³H]GABA from [³H]GLU as well as on the uptake of [³H]GLU. The application of high potassium (60 mM) stimulation exhibited a significant enhancement of the release of this newly synthesized [³H]GABA in a Ca²⁺ dependent manner. Kinetic analysis revealed that the evoked release of newly synthesized [³H]GABA was approximately two times greater than that of previously-loaded [³H]GABA, whereas no significant difference was observed in the spontaneous release. An immobilization stress in water failed to affect the release of newly synthesized [³H]GABA from striatal slices despite the occurrence of a significant enhancement of GABA formation in this structure.These results suggest that newly synthesized GABA may be preferentially released from its nerve terminals in response to the excitation of neurons at least in the striatum as compared with previously accumulated GABA.     

Decreased [3H]serotonin and [3H]spiperone binding consequent to lipid peroxidation in rat cortical membranes

The effect of lipid peroxidation on two types of serotonin binding sites was investigated. Incubation of rat cortical membranes with an ascobate-dependent peroxidizing system resulted in the formation of malonyldialdehyde and significant decreases in the specific binding of [³H]serotonin and [³H]spiperone to the treated membranes. When ascorbate concentrations were varied from 0.025 to 6.0 mM, malonyldialdehyde production increased to a maximum at 0.5 mM ascorbate and then declined. Conversely, the specific binding of [³H]serotonin and [³H]spiperone decreased to a minimum at 0.5 mM ascorbate and then increased. Regression analysis of the data revealed that the decrease in the two binding sites was linearly correlated to lipid peroxidation.     

2-Nitroimipramine: A selective irreversible inhibitor of [3H] serotonin uptake and [3H] imipramine binding in platelets

The effect(s) of a new imipramine analogue, 2-nitroimipramine, on high affinity [³H] imipramine binding and [³H] serotonin uptake in human platelets were studied. 2-Nitroimipramine was found not only to be a very potent inhibitor of [³H] imipramine binding and [³H] serotonin uptake but was found to irreversibly inhibit binding and uptake simultaneously. This finding supports previous observations from our laboratory and others that high affinity imipramine binding labels serotonin uptake or transport sites. 2-Nitroimipramine should prove an important tool for subsequent studies of the molecular mechanism(s) involved in the transport of serotonin and the binding of imipramine to platelet and brain membranes.     

Light microscopic autoradiographic visualization of [3H]-arginine vasopressin binding sites in rat brain

Specific [³H]-arginine vasopressin ([³H]-AVP) binding sites were identified in the rat brain by light microscopic autoradiography. Discrete intrahypothalamic nuclei were densely labelled by [³H]-AVP. High specific binding was observed in the paraventricular and supraoptic nuclei. These binding sites may represent specific receptors for AVP, postulated to exist in the mammalian central nervous system.     

The incorporation of [3H]glucosamine, [3H]fucose and [14C]mannose into protein in the rat anterior pituitary incubated in vitro

Rat anterior hemipituitaries incubated in vitro rapidly take up and incorporate into protein D-[6-³H]-glucosamine · HCl, D-[1-¹⁴C]mannose and L-[G-³H]fucose. The newly labeled protein was only slowly released into a Krebs-Ringer bicarbonate incubation medium. Glucosamine- or mannose-labeled protein was barely detectable in the medium after a 30–60 min incubation whereas about 4% of all fucose-labeled protein had already been released into the incubation medium by 30 min. Puromycin · 2HCl (1 mM) inhibited incorporation of glucosamine or mannose into protein to 40% or less of control values within 30 min; fucose incorporation was not significantly inhibited before 45 min. Acid hydrolysis followed by amino acid analysis of glucosamine-labeled protein yielded significant amounts of label in glucosamine, galactosamine and apparent glucosamine-degradation products but no significant amount of label in any amino acid.     

[3H]muscimol binding in the rat retina

Crude membrane fractions were prepared from rat retinae and used to study the specific binding of [³H]muscimol, a potent GABA agonist. Specific [³H]muscimol binding was enhanced 2–3 fold by pretreatment of the membranes with 0.025% Triton X-100. Two muscimol binding sites were demonstrated with K_D values of 4.4 and 12.3 nM. GABA, muscimol, and 3-aminopropanesulfonic acid were the most potent inhibitors of specific [³H]muscimol binding with K_I values of 15, 10, and 50 nM, respectively. These data are consistent with binding to the synaptic GABA receptor.     

Direct photoaffinity labeling of the primary region of the ouabain binding site of (Na+ + K+)-ATPase with [3H]ouabain, [3H]digitoxin and [3H]digitoxigenin

The tritiated cardiotonic steroids, ouabain, digitoxin, and digitoxigenin are shown to photolabel the large polypeptide but not the glycoprotein or proteolipid component of the (Na⁺ + K⁺)-ATPase when they are bound to the inhibitory site and exposed to light of 220 or 254 nm. The extent of photolabeling is low, less than 1%, and is limited by photocross-linking of the enzyme. The mechanism of photoincorporation does not appear to be either photolysis of the lactone ring in ouabain or photolysis of tryptophan or tyrosine residues in the polypeptide.     

Uptake of [3H]-arachidonic acid by human endometrium. Differences between normal and menorrhagic tissue

Human proliferative and secretory endometrium from normal women and from menorrhagic patients was maintained in culture for up to a 24 h in the presence of [³H]-arachidonic acid (³H-AA). This prostaglandin (PG) precursor was incorporated into endometrial neutral lipids and phospholipids in a time-dependent manner. Uptake of ³H-AA into phospholipids was significantly higher in normal secretory endometrium than in normal proliferative endometrium. However, this increased uptake of ³H-AA into phospholipids between the 2 phases of the cycle did not occur in menorrhagic endometrium. In contrast, uptake of ³H-AA into neutral lipids (especially triglyceride) was approximately 2-fold higher in menorrhagic endometrium compared to normal endometrium at both stages of the cycle, particularly during the proliferative phase. Abnormalities apparently exist in menorrhagic endometrium in the uptake processes which control arachidonic acid (AA) turnover. These abnormalities may be responsible, in part for abnormal PG production by menorrhagic endometrium.     

Subcellular distribution of [3H]-prostaglandin E2 binding sites in porcine gastric mucosa

The distribution of PGE₂ binding sites in four subcellular fractions (F1–F4) from porcine fundic mucosa obtained by gradient centrifugation was examined. Binding of HPGE₂ to fractions F2–F4 was specific, dissociable, saturable and pH dependent. A significant degree of specific binding was not evident in F1. The Scatchard analysis of binding to F2 and F3 revealed heterogenous populations of binding sites with similar dissociation constants but greater concentrations of binding sites than was evident in the initial 30,000 xg homogenate protein. A single class of low affinity binding sites was evident in F4. The ratio of total: nonspecific binding was approximately equal in F2 and F3. The ratio was considerably smaller in F4. The activity of 5' nucleotidase the marker enzyme for plasma membranes followed this ratio. There was no correlation between the binding ratio and marker enzyme activities for mitochondrial membranes and endoplasmic reticulum. These data suggest that high affinity PGE₂ binding sites occur predominantly on the plasma membrane from gastric mucosal tissue.     

Effect of phencyclidine on [3H]QNB binding

Intraperitoneal injection of phencyclidine before intravenous injection of [³H] Quinuclidinyl benzilate (QNE, 1.6 μg/kg) significantly increased the amount of radioactivity found in the brains of female C57BL/6J mice one hour after the ³H-QNB administration. This effect was found in hypothalamus, cortex, hippocampus and striatum and was decreased by pretreatment of the animal with atropine. The magnitude of the enhancement varied as a function of dose but did not change across the time span studied. These data are in contrast to our findings and those of others of inhibition of the specific binding of ³H-QNB to muscarinic cholinergic receptors by PCP $\text{in vitro}$. When atropine or PCP was administered $\text{in vivo}$ and the tissue later analyzed $\text{in vitro}$, no effects of the drugs were observed on ³H-QNB binding. The reasons for the differences remain a matter of speculation.     

Beta-adrenergic receptors in early human placenta characterization of [3H]-dihydroalprenolol binding

The binding characteristics of beta-adrenergic ligand [³H]-dihydroalprenolol (DHA) were determined in particulate membranes of early human placenta (8 – 12 weeks of gestation). [³H]-DHA binding to crude membrane fractions was rapid, reversible, saturable and linearly correlated with the membrane protein concentration. Scatchard analysis of saturation experiments showed a K_D of 2.80 ± 0.9 nM and a density of binding sites of 330.30 ± 93.5 fmol/mg protein. Agonist potency isoproterenol epinephrine norepinephrine indicated that early human placenta contains an adrenergic receptor of beta-2 subtype.     

Light microscopic autoradiographic localization of [3H] substance P binding sites in rat thoracic spinal cord

We have used light microscopic autoradiography to look for the distribution of [³H] substance P receptors in the thoracic spinal cord of the rat. High densities of autoradiographic grains were localized to the intermedialateral cell column, the central canal and the substantia gelatinosa of the dorsal horn.