Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or chaning the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5′-(N-ethylcar☐amido)adenosine (NECA, 10 μM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 ± 0.02 (S.E.) to 1.80 ± 0.02 (p < 0.001) while inosine (10 μM), a poor adenosine receptor agonist, had no effect (1.73 ± 0.04, p =n.s. vs. control, p < 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA > adenosine = N⁶-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 μM) did not significantly change the number or affinity of [³H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.     

Voltage-dependent channels permeable to K+ and Na+ in the membrane ofAcer pseudoplatanus vacuoles

Summary The patch-clamp technique in whole-cell configuration was used to study the electrical properties of the tonoplast in isolated vacuoles fromAcer pseudoplatanus cultured cells. In symmetrical KCl or K₂ malate solutions, voltage- and time-dependent inward currents were elicited by hyperpolarizing the tonoplast (inside negative), while in the positive range of potential the conductance was very small. The specific conductance of the tonoplast at –100 mV, in 100mm symmetrical KCl was about 160 S/cm². The reversal potentials (E _rev) of the current, measured in symmetrical or asymmetrical ion concentrations (cation, anion or both) were very close to the values of the K⁺ equilibrium potential. Experiments performed in symmetrical or asymmetrical NaCl indicate that Na⁺ too can flow through the channels. NeitherE _rev nor amplitude and kinetics of the current changed by replacing NaCl with KCl in the external solution. These results indicate the presence of hyperpolarization-activated channels in tonoplasts, which are permeable to K⁺ as well as to Na⁺. Anions such as Cl^– or malate seem to contribute little to the channel current.     

The chemical characterization of melanin contained in substantia nigra of human brain

The pigment of substantia nigra human brain has been extracted by a mild procedure consisting of washes with phosphate buffer, methanol and incubation with SDS-proteinase. Pyrolisis gas chromatography mass spectrometry infrared spectrometry, termogravimetric analysis and elemental analysis were the techniques used for the chemical characterization. An indole moiety bound to a sulfur containing amino acid and to palmitic acid were the main aspects found in the structure. The presence of a 7% inorganic component was observed. This probably contains Fe, Cu, Zn and Cr which are also relevant, for the formation and the role of melanin in substantia nigra neurons. The fatty acid moiety is chemically bound to the indole structure as it was not eliminated by repeated methanol washing. The same situation occurs for the sulfur containing gropu. Considering these data and the most abundant molecules present in substantia nigra the precursor of neuromelanin seems to be a cysteinyl-cethecol, to which is then bound a palmityl group.     

Selection and characterization of transferrin receptor mutants using receptor-specific antibodies

Lymphoma cell lines were selected by growth in transferrin receptor-specific antibodies and in transferrin receptor-specific antibody coupled to ricin toxin. Sequential selections were used to isolate lines with multiple mutations affecting the transferrin receptor molecule. Mutant cell lines were characterized by their growth in antibody and their antibody-binding properties. Two basic types of mutations were found. One type resulted in the loss of a binding determinant for the antibody used for selection on one of the two transferrin receptor allelic products. The other type of mutation resulted in the loss of cell-surface expression of the entire gene product of one of the transferrin receptor alleles.     

Relative Stability of Pertussis Vaccine Preserved with Merthiolate, Benzethonium Chloride, or the Parabens

When stored at 4 C, or heated at 22 or 35 C followed by storage at 4 C, the potency of pertussis vaccines preserved with Merthiolate was more stable than the potency of vaccines preserved with benzethonium chloride or the parabens (methyl- and propyl-p-hydroxybenzoate). Without preservative, potency was more stable than in the presence of benzethonium chloride or the parabens, but less stable than when Merthiolate was present. The histamine-sensitizing factor of the vaccines likewise decreased with the loss of potency. The deleterious effect of benzethonium chloride and the absence of the stabilizing effect of Merthiolate were contributing factors, if not the sole cause, for the instability of pertussis vaccine in quadruple antigen vaccine (diphtheria and tetanus toxoids and pertussis and poliomyelitis vaccines).     

Aspects of neural plasticity in the central nervous system—III. Methodological studies on the microdialysis technique

Some methodological aspects of the intracerebral microdialysis technique have been investigated: the existence of a pressure gradient at the level of the dialyzing membrane, the substance diffusion from the microdialysis probe and the extent of tissue damage induced by the implantation of the microdialysis probe. At the level of the dialyzing membrane a rough balance between the pressure inside the probe and the one present in the extracellular fluid compartment has been observed. The pattern of substance diffusion in the tissue showed a large variability depending on the substance used and the experimental conditions. Relevant deductions can be made by the use of labeled markers. By means of this approach, the diffusion pattern of tritiated ganglioside GM1 in the tissue around the probe could be shown to follow a biexponential pattern, suggesting a two-step process of diffusion. The degree of tissue damage induced by the microdialysis probe was assessed by analyzing the glial reaction, and was measured by means of semiquantitative immunocytochemistry of glial fibrillary acidic protein immunoreactivity. Only a limited area of neuronal damage was observed in the region surrounding the microdialysis probe. The amount of glial reaction after probe implantation was shown to be comparable with that induced by the implantation of a microinjection cannula.