Effect of amphotericin B on membranes: a spin probe study

The effect of the polyene antibiotic amphotericin B on the electron paramagnetic resonance spectra of lipid probes intercalated in model membranes was examined. When the antibiotic was added to the aqueous phase, no spectral effects occurred. However, when the antibiotic was incorporated during membrane preparation, changes in spectral parameters suggested the appearance of a new phase. The spectral changes do not necessarily corroborate the pore models proposed previously for amphotericin B in membranes. With a spin probe that partitions between water and membrane, an interaction between the amphotericin B and probe is observed. This interaction does not occur in the membrane, but in the aqueous phase, between the probe and the aggregated antibiotic. Some of the equilibria involving the antibiotic appear to be achieved slowly.     

A possible involvement of cyclic AMP in the expression of desensitization of the nicotinic acetylcholine receptor. A study with forskolin and its analogs

Forskolin, an activator of adenylate cyclase, and its analogs were studied on the nicotinic acetylcholine receptor-ion channel complex (AChR) of rat and frog skeletal muscles. At nanomolar concentrations, forskolin caused desensitization of the AChR located at the junctional region of innervated and the extrajunctional region of chronically denervated rat soleus muscles. The desensitization of the AChR occurred without alteration of the conducting state (channel lifetime, conductance or bursting) as shown by single channel currents. Accordingly, forskolin decreased the peak amplitude of the repetitive evoked endplate currents in frog sartorius muscles. These findings taken together with the good correlation found between the effects of forskolin and its analogs on the desensitization of the nicotinic AChR and their ability to activate adenylate cyclase suggested a possible involvement of phosphorylation of AChR via cyclic AMP on the desensitization process.     

Spin label reduction kinetics,a procedure to study the effect of drugs on membrane permeability: The effects of monosodium urate,dimethyl sulfoxide and amphotericin B

A method is presented to study the effect of drugs on membrane permeability. It is based on the reduction of a spin label trapped in the internal aqueous compartment(s) of membranes by ascorbate ions added to the bulk aqueous phase. The decay of the electron spin resonance signal of the spin label as a function of time gives an indication of the effect of added agents on the permeability of membranes. To demonstrate the technique, the effect on model membranes of egg phosphatidylcholine of the gout-implicated compound monosodium urate, the aprotic solvent dimethyl sulfoxide and the polyene antibiotic amphotericin B were examined. Monosodium urate did not affect the permeability, casting doubt on a proposed mechanism whereby the agent disrupts the membranes via hydrogen bonding. Dimethyl sulfoxide promoted a gradual increase in rate of solute passage across cholesterol-containing model membranes. Amphotericin B had pronounced effect on the permeability of cholesterol-containing membranes, causing nearly total loss of paramagnetism immediately after addition. Some aspects of the mechanism of action of the drugs are discussed as well as the advantages and disadvantages of the method. The experiments also allow the evaluation of the effect of surface charge and cholesterol on the dimensions of model membranes.     

Effects of amphotericin B on membrane permeability--kinetics of spin probe reduction

The effect of the polyene antibiotic amphotericin B on the permeability of both unilamellar and multilamellar model membranes is investigated. The method measures the loss of the electron paramagnetic resonance signal of a spin probe, trapped in the aqueous compartment of a lipid dispersion, upon addition of ascorbate ions to the bulk aqueous phase. Amphotericin B causes large increases in the permeability of cholesterol-containing egg phosphatidylcholine membranes, whereas the effects are small in the absence of sterol and do not depend on surface charge. The effect of amphotericin depends upon the antibiotic:sterol mole ratio. The antibiotic appears to be unable to cross the membrane, acting only on the outermost bilayer of a multibilayer dispersion. When a phospholipid in the gel phase is used, amphotericin B causes large increases in permeability, independently of the presence or absence of sterol. It is suggested that the mechanism of action of amphotericin B is different for lipids in the liquid crystalline or gel states.     

Nicotinic acetylcholine receptors in cultured neurons from the hippocampus and brain stem of the rat characterized by single channel recording

Single channel recording techniques have been applied to neurons cultured from the hippocampus and the respiratory area of the brain stem of fetal rats in order to search for nicotinic acetylcholine receptors (nAChR) in the central nervous system. In addition to acetylcholine (ACh), the potent and specific agonist (+)-anatoxin-a was also used to characterize nicotinic channels. nAChRs were concentrated on the somal surface near the base of the apical dendrite, and in some patches their density was sufficient to record 2 or more channel openings simultaneously. Although a multiplicity of conductance states was also evident, the predominant population showed a single channel conductance of 20 pS at 10 degrees C. Thus, these neuronal nAChRs resembled the embryonic or denervated-type nAChRs in muscle. However, channel opening and closing kinetics were faster than reported for similar conductance channels in muscle. Therefore the nicotinic channels described here are similar but not identical to those of the well-characterized muscle nAChR, in agreement with biochemical, pharmacological, and molecular genetic studies on brain AChR.