The ubiquitous localization of type I hexokinase in rat peripheral nerves, smooth muscle cells and epithelial cells

Summary An indirect immunoperoxidase technique has been used to determine the localization of type I hexokinase in a wide variety of Carnoy-fixed, paraffin-embedded rat tissues. The results suggest that the widespread tissue distribution of the isoenzyme is due to its ubiquitous localization in the nervous, smooth muscle and epithelial components of each tissue. The majority of the immunostaining was confined to cells with substantial energy requirements which are probably mainly satisfied through the breakdown of glucose. This observation is consistent with the known predominance of type I hexokinase in the central nervous system and with the regulatory role allotted to it in this tissue.     

Histochemical and immunohistochemical localization of hexokinase isoenzymes in normal rat liver

Summary Histochemical and immunohistochemical procedures have been used to examine the localization of three of the four hexokinase isoenzymes present in the liver of fed female Wistar rats. Distinctive distribution patterns were found for hexokinase type I and glucokinase but hexokinase type II was not detectable. Hexokinase type I was identified in sinusoidal cells and in bile duct epithelia, nerves and arteries in the portal triad. Glucokinase, the major isoenzyme, was confined to parenchymal cells where it was present in much higher amounts in perivenous compared with periportal hepatocytes. Staining within these two zones was not homogeneous and each had a mosaic appearance caused by the presence of a few hepatocytes containing little or no glucokinase amongst the majority of darkly stained cells in perivenous areas and a few darkly stained cells amongst the majority of unstained cells in periportal areas. Hence, hepatocytesin situ are a strikingly heterogeneous population of cells. Their metabolic status cannot be controlled simply by the differential supply of oxygen, substrates and hormones to different regions of the liver acini as proposed in the metabolic zonation model. Phenotypic differences may exist between cells within a given metabolic zone which influence their ability to respond to different environmental conditions.     

Mechanisms of serotonin-induced lymphocyte proliferation inhibition

When human peripheral blood lymphocytes were stimulated with phytohemagglutinin in the presence of serotonin, inhibition of [3H]thymidine incorporation occurred, the most marked inhibition occurring at high (10(-3)M) serotonin concentrations. This effect could not be reversed by the addition of Interleukin 2 (IL-2)-containing supernatants. Cytofluorometric analysis showed that virtually all of the cells remained in the G0 phase (unactivated) at 24 hr while some of the cells entered the G1a and G1b phases of the cell cycle by 42 hr. The cellular production of IL-2 was not affected by serotonin, as supernatants of treated cultures contained essentially the same IL-2 titers as did control cultures. Serotonin seemed to primarily affect cell activation and had little or no effect on proliferating cells. This was further confirmed by the lack of effects of serotonin on a variety of established proliferating lymphocyte, macrophage, and fibroblast cell lines. By contrast, dose-dependent inhibition of IL-2-dependent CTLL cells occurred. Serotonin was not toxic even at 10(-3) M concentrations. A marked decrease in IL-2 receptors and a change in their distribution on responder cells was seen when treated cultures were examined with the anti-Tac monoclonal antibody. At 24 hr this effect was contrastingly not seen for the OKT-8 marker, although a slight decrease in OKT-4-positive cells was seen. Serotonin thus produced an inhibition of lectin-stimulated lymphocyte proliferation via a mechanism independent of IL-2 production, and caused a decrease in the expression and distribution of IL-2 receptors on the surface of responder cells.     

The effect of short-term fluctuations in pH on NO−3 uptake and intracellular constituents in Skeletonemacostatum (Grev.) Cleve

Measurements of uptake rates, intracellular nitrogen pools, and other key intracellular constituents were made during exponential growth in Skeletonema costatum (Grev.) Cleve under varying pH levels. An understanding of the overall effects of extracellular pH on the above mentioned cellular parameters is crucial in order to ascertain the degree to which pH must be regulated and monitored in laboratory experiments with marine phytoplankton.It was found that uptake rates and intracellular pool sizes of NO^?₃ were directly influenced by the extracellular pH level, whereas, other cellular compounds remained relatively unchanged. Therefore, nitrogen uptake and intracellular nitrogen storage are dependent on key H⁺ and OH_? ion transport mechanisms that are associated with phytoplankton metabolism. These findings reiterate the fact that investigators examining nitrogen uptake and assimilatory mechanisms in marine phytoplankton must be conscious of cellular H ⁺ and OH^? fluxes that contribute to intracellular pH regulation and changes in extracellular pH levels, both of which interact to affect phytoplankton metabolic processes.     

Ionic Regulation of the Binding of dl-[3H]2-Amino-4-Phosphonobutyrate to l-Glutamate-Sensitive Sites on Rat Brain Membranes

Abstract: The effects of ions on the binding of the excitatory amino acid analogue dl -[³H]2-amino-4-phosphon-obutyrate to l -glutamate-sensitive sites on rat brain synaptic membranes was investigated. The divalent cations manganese, magnesium, strontium, and particularly calcium, produced a marked enhancement in specific binding. However, this effect was manifest only in the presence of added chloride, or to a lesser extent, with bromide ions. Application of saturation analysis revealed that both chloride and calcium acted to increase the binding site density in a concentration-dependent manner, without affecting the dissociation constant. The only other ionic species found to have a significant effect on 2-amino-4-phosphonobutyrate binding was sodium, which produced an apparent reduction in site affinity, without modifying the binding site density. Although the significance of these striking ionic effects is as yet unknown, it seems feasible that chloride (and possibly also calcium) ions may serve a role in regulating the interaction of excitatory amino acids with their physiological receptors.     

pH Selectivity of N-Ethylmaleimide Reactions with Opiate Receptor Complexes in Rat Brain Membranes

N-Ethylmaleimide (NEM) decreases opiate agonist binding presumably by blocking crucial sulfhydryl (SH) groups at receptor binding sites. At physiological pH, NEM decreased GTP and manganese regulation but increased sodium effects on [3H]D-Ala2-Met5-enkephalinamide (D-Ala enk) binding to rat brain membranes. To determine the apparent pK values of putative SH groups in opiate receptors that react with NEM, rat brain membranes were incubated with 100-250 microM NEM in buffers ranging from pH 4.5 to 8.0. Results showed that lowering pH below 6.5 reduced the NEM effect on opiate receptor functions and that the apparent pK values of NEM-reacting SH groups in binding and regulatory sites ranged between 5.4 to 6.0. Most of the total SH groups in brain membranes continued to react with NEM at low pH, so that when nonspecific SH groups were blocked by incubating membranes at pH 4.5 with NEM, opiate receptors became sensitive to very low concentrations (1 microM) of NEM.     

Mechanism of A23187-induced airway obstruction in the guinea pig

Exposure of conscious guinea pigs to A23187 aerosol produced a concentration-related increase of excised lung gas volume (ELGV), . ., postmortem pulmonary gas trapping. Measurements of ELGV were highly correlated with measurements of dynamic compliance (C_dyn) and total pulmonary resistance (R_L) and were used as an indication of airway obstruction. We pretreated guinea pigs intravenously with the following drugs: atropine; LY163443, a selective LTD₄/E₄ antagonist; indomethacin; propranolol; and pyrilamine. The guinea pigs were exposed for 8 minutes to the A23187 aerosol, and ELGV measurements were then made. Atropine or pyrilamine prevented the A23187-induced gas trapping. Indomethacin or propranolol tended to potentiate the response and when combined, they potentiated the gas trapping by 80%. LY163443 had no effect alone, but when combined with indomethacin, propranolol, and pyrilamine, inhibited A23187-induced gas trapping by 67%. We conclude that cholinergic and histaminergic mechanisms play major roles in the ionophore-induced pulmonary gas trapping of the guinea pig. With appropriate pretreatment, sulfidopeptide leukotrienes may produce a substantial effect.     

Effect of urethan anesthesia on cigarette smoke-induced airway injury in guinea pigs

The effect of urethan anesthesia on cigarette smoke-induced airway responsiveness and permeability was studied in the guinea pig. Airway responsiveness was determined by measuring changes to airway resistance to graded doses of aerosolized histamine, and mucosal permeability was determined by measuring the appearance of fluorescein isothiocyanate-dextran (FITC-D) in the blood and examining its distribution in lung tissue after it had been delivered to the lung in an aerosol. The results confirm previous studies that smoke exposure increased airway responsiveness and mucosal permeability. They also show that urethan anesthesia administered before smoke exposure prevented the smoke-related changes in airway reactivity and mucosal permeability. In animals that remained conscious during the smoke exposure, there was increased deposition of the dextran in the regions of the bronchioloalveolar junctions with a more rapid uptake of FITC-D into the blood. We postulate that, when urethan anesthesia is administered before smoke exposure, the exudative phase of the inflammatory reaction produced by smoke exposure is suppressed.