Reversal of acetylcholine potentials in eel electroplaque.

Although the eel electroplaque is a major source of purified acetylcholine (ACh) receptor, the electrophysiological properties of the receptor have not been studied in detail. In particular, the reversal potential for the action of ACh on the postsynaptic membrane has not been measured directly. In order to obtain the reversal potential, ACh was applied iontophoretically from a micropipette onto the innervated membrane. The resulting depolarization (ACh potential) decreased in amplitude as the cell was depolarized, reached zero at a reversal potential of about -4 millivolts, and then reversed in sign as the inside of the cell was made increasingly more postive. The relation between ACh potential amplitude and membrane potential was nonlinear because of a decrease with depolarization of the peak conductance change produced by the drug.

Effect of acetylcholine on postjunctional membrane permeability in eel electroplaque.

Acetylcholine (ACh) was applied iontophoretically to the innervated face of isolated eel electroplaques while the membrane potential was being recorded intracellularly. At the resting potential (about -85 mV) application of the drug produced depolarizations (ACh potentials) of 20 mV or more which became smaller when the membrane was depolarized and reversed in polarity at about zero membrane potential. The reversal potential shifted in the negative direction when external Na+ was partially replaced by glucosamine. Increasing external K+ caused a shift of reversal potential in the positive direction. It was concluded that ACh increased the permeability of the postjunctional membrane to both ions. Replacement of Cl- by propionate had no effect on the reversal potential. In Na+-free solution containing glucosamine the reversal potential was positive to the resting potential, suggesting that ACh increased the permeability to glucosamine. Addition of Ca++ resulted in a still more positive reversal potential, indicating an increased permeability to Ca++ as well. Analysis of the results indicated that the increases in permeability of the postjunctional membrane to K+, Na+, Ca++, and glucosamine were in the ratios of approximately 1.0:0.9:0.7:0.2, respectively. With these permeability ratios, all of the observed shifts in reversal potential with changes in external ionic composition were predicted accurately by the constant field equation.

Multiple neuropeptides exert a direct effect on the same isolated single smooth muscle cell.

The contractile effect of various neuropeptides was examined by pressure ejecting these agents from a pipette onto single smooth muscle cells freshly dissociated from the stomach of Bufo marinus. Substance P, cholecystokinin-octapeptide, and bombesin caused contraction, whereas vasoactive intestinal peptide, secretin, and dopamine inhibited acetylcholine-induced contractions. Acetylcholine and the three peptides which produced contraction were found in some instances to act on the same cell, suggesting that receptors for these agents exist on one and the same cell.

Artifacts associated with quick-freezing and freeze-drying.

We have studied the structures produced when nonbiological samples were subjected to quick-freezing and freeze-drying with a liquid helium cooled freeze-slamming device. Samples examined in this way included sodium chloride, sucrose, and Tris buffer. A variety of filamentlike and trabeculumlike structures were formed in these preparations. These structures may represent eutectic mixtures formed during the growth of small ice crystals during the freezing process, and exposed during the rapid sublimation of pure ice during the etching process. Samples of biological membranes (isolated chloroplast membranes) were prepared in various buffers by means of this technique. In distilled water, excellent replicas of membrane surfaces were obtained. In salt solutions, however, the membranes appeared to be embedded in a network of thin filaments appearing very much like a cytoskeletal lattice. It is concluded that extreme caution must be used when employing this preparation technique for studies of cell architecture, and that extensive washing of cell components in distilled water may be necessary to obtain faithful representations of cell structure.