ELMQVIST, D. and J.-0. JOSEFSSON. The nature of the neuromuscular blockproduced by neomycine. Acta physiol. scand. 1962. 54. 105-110. -The neuromuscular block produced by neomycine has been studied in the rat diaphragm and the frog sartorius by means of intra-and extracellular recording techniques. The block is due to a reduction in the amplitude of the end-plate potential. This reduction is partly caused by a neomycine induced decrease in the sensitivity of the postjunctional end-plate membrane to the depolarizing action of acetylcholine. Neomycine reduces the amount of transmitter reIeased from the motor nerve in response to a nerve volley or to a high external potassium concentration. This prejunctional effect of neomycine is antagonized by an excess of Ca ions. Neomycine produces as shown by PITTINGER and LONG (1 958) and CORRADO et al. (1958) a neuromuscular block in vivo as well as in vitro. This block is antagonized by neostigmine and calcium. Neostigmine is effective only when the block is partial but calcium is effective even when neomycine has caused a complete neuromuscular block.The experiments of CORRADO et al. indicated that the neuromuscular block caused by neomycine differed from that produced by competitive blocking agents as curare and that produced by drugs which depolarize the end-plate as decamethonium. They suggest the possibility that neomycine has a presynaptic site of action similar to that of high magnesium concentrations (ENGBAEK 1952, DEL CASTILLO and ENGBAEK 1954).This uncertainty regarding the site of action of neomycine at the neuromuscular junction prompted us to investigate its effect on transmitter release and on the chemical sensitivity of the postjunctional membrane. 105

Pinocytosis induced by different cations was studied by phase contrast in Amoeba proteus. Monovalent cations were more effective inducers the smaller the hydrated size of the ion (Cs>K>Na>Li). Divalent cations were less active inducers than monovalent ions and among the divalent ions tested calcium was unable to induce measurable pinocytosis. Tris+ possessed properties as an inducer similar to sodium although the intensity of Tris‐induced pinocytosis was greater. The sensitivity to the inducing cation decreased in proportion to the concentrations of calcium and hydrogen ions in the medium. Addition of a calcium chelating agent to the inducer decreased the maximum number of channels observed. Among the divalent cations present in the culture solution only calcium could restore the pinocytosis intensity to normal. The receptors in the cell membrane upon which inducing ions act are proposed to be negatively charged groups, the ionization of which varies with pH. Their availability to inducers is diminished when the calcium concentration of the medium is increased. A minimal calcium concentration is, however, required for a normal function of the channel forming system. The differene in activity between potassium and sodium in the induction of pinocytosis might be a factor controlling the intracellular content of these ions in the amoeba. The outward diffusion of potassium from the cell might explain the phenomenon of “spontaneous” pinocytosis.

JOSEFSSON, J.-0. and S. THESLEFF. Electromyographic jindings in experimental botulinum intoxication. Acta physiol. scand. 1961. 51. 163-168. -

A characteristic feature of induced pinocytosis in Amoeba proteus is the formation of broad channels by invagination of the cell membrane. This process, which requires Ca2+, occurs in response to depolarising cations. High Ca2+ levels reduce pinocytosis induced by cations such as Na+ and Tris+, whereas pinocytosis induced by K+ is less affected by Ca2+ (ref. 4). Agents which interfere with the calcium metabolism of the amoeba will therefore either stimulate or inhibit pinocytosis induced by Na+ (ref. 5). Among the agents which are supposed to reduce Ca2+ influx across cell membranes or otherwise decrease cellular availability of Ca2+ are the opiates and opioid peptides, high doses of which have been reported to affect the amoeba. Accordingly, Met-enkephalin, morphine and codeine potentiate the inhibition of pinocytosis caused by Ca2+-binding agents and reverse the calcium blockade of pinocytosis mediated by caffeine. In this report we show that pinocytosis induced by Na+ or Tris+ is suppressed by beta-endorphin, Metenkephalin and morphine. These effects were abolished or diminished by an opiate receptor antagonist, (-)naloxone, by increasing the Na+ concentration, or by addition of Ca2+.

An investigation of the relationship between the polarized state of the membrane and the onset and the intensity of pinocytosis was made in Amoeba proteus. Membrane potential and input resistance was in all instances found to decrease in approximate proportion to the number of channels when pinocytosis was induced by a variety of alkali metal ions at varying pH. Channels began to appear when the membrane was depolarized to -30 mV by the inducer of pinocytosis. With all inducers the maximum pinocytosis was encountered at membrane potentials close to zero. No positive potentials were recorded when the chloride salts of the inducing cations were used. At high concentrations of alkali ions a transient increase of the chloride permeability caused short-lasting hyperolarizations of the membrane. Inhibition of pinocytosis by Ca++ was accompanied by an increase of input resistance and membrane potential. The selectivity of the membrane to different alkali metal ions observed as changes in pinocytosis intensity, membrane potential and input resistance was found to vary with the concentration of the inducer and with the Ca++ concentration of the extracellular solution. Displacement of membrane bound Ca++ appeared to decrease the field strength of charged groups in the membrane altering its selectivity among alkali cations. The formation of pinocytotic channels is suggested to require translocation of Ca++ from the membrane into the cell and would therefore be closely related to the electrical properties of the amoeba.