The actions of verapamil at the neuromuscular junction.

1. The actions of the calcium channel blocker verapamil were studied at the neuromuscular junction of the frog Rana pipiens. 2. In the presence of 50 microM verapamil, subthreshold endplate potentials were produced, and the quantal content was reduced by a factor of 3. 3. Verapamil (10-50 microM) also reduced the postjunctional membrane sensitivity as measured by (a) carbachol iontophoresis and (b) miniature endplate potential amplitude. In addition, verapamil had a strong inhibitory effect on the postjunctional membrane response to repetitive iontophoretic application of carbachol. 4. Thus, verapamil has both pre- and postsynaptic actions at the neuromuscular junction.

Effects of chronic prednisolone treatment on postjunctional membrane responses to agonist.

Using frogs treated 1 month with prednisolone, we studied responses of the muscle postjunctional membrane to acetylcholine and carbamylcholine. In control muscles following a bath application of 3 microM neostigmine or 30 microM pyridostigmine, there was an increased depolarization response to iontophoretically applied acetylcholine or carbamylcholine. Results with prednisolone-treated frog muscle were essentially identical. In some fibers of prednisolone-treated muscle there was a decrementing response to repetitive iontophoretic application of carbamylcholine. Prolonged administration of prednisolone may accelerate desensitization of acetylcholine receptors during repetitive application of agonist.

Ruthenium red reduces acetylcholine sensitivity and increases desensitization at the frog neuromuscular junction.

The actions of Ruthenium Red on the synaptic membrane were studied at the neuromuscular junction of the frog Rana pipiens. Ruthenium Red blocks mitochondrial calcium transport and thus is expected to elevate the intracellular calcium level [Alnaes and Rahamimoff (1975) J. Physiol., Lond. 248, 285-306]. The postjunctional membrane sensitivity was reduced by Ruthenium Red as determined by (a) the amplitude of the miniature endplate potentials, (b) iontophoretic application of carbachol, (c) microperfusion of carbachol. Desensitization was assayed by measuring the decline in the amplitude of the postsynaptic depolarization produced during repetitive iontophoretic application of carbachol. Following Ruthenium Red treatment desensitization was increased. This action of Ruthenium Red was enhanced by raising the extracellular calcium concentration from 1.8 to 10 mM.

Action of caffeine in excitation-contraction coupling of frog skeletal muscle fibres.

1. Frog sartorius muscle bathed in 1 mM-caffeine generates brief asynchronous contraction of individual sarcomeres, 'sarcomeric oscillations', and propagated contractile waves. 2. Analysis of cinematographic records shows that during sarcomeric oscillations the sarcomere length decreases and the distribution of sarcomere lengths is wider than in controls. 3. Caffeine can produce sarcomeric oscillations in K depolarized muscle fibres and, to a limited extent, in glycerol-treated muscle fibres. 4. Treatment of muscle with dantrolene sodium blocks production of sarcomeric oscillations by caffeine. 5. In caffeine-treated muscle fibres, electrically produced depolarization could initiate or increase the frequency of sarcomeric oscillations, and electrical hyperpolarization diminishes the frequency or stops sarcomeric oscillations. 6. Caffeine solution bathing a muscle undergoing sarcomeric oscillations (the perfusate), when applied to a fresh muscle, initiates sarcomeric oscillations with a relatively short latency. 7. An U.V.-absorbance peak at 245 nm develops in the caffeine solution bathing a muscle undergoing sarcomeric oscillations. 8. It was found that a contraction-regulating substance (oscillogen) is released from a muscle undergoing sarcomeric oscillations. From results of selective dialysis and gel permeation chromatography, the molecular weight of the oscillogen is estimated to be between 700-1000 daltons. 9. It is proposed that the oscillogen is a normally occurring substance which functions in excitation-contraction coupling at the T-tubule terminal cistern junction in skeletal muscle.

Factors in the production of experimental autoimmune myasthenia gravis in acetylcholine receptor immunized rabbits.

The development of experimental autoimmune myasthenia gravis (EAMG) was studied in 10 rabbits which were repeatedly injected with Torpedo acetylcholine receptor (AChR). Serum samples were obtained at various times for determination of complement fixing antibody level, serum complement level and the capacity of serum to inhibit neuromuscular transmission in amphibian muscle (passive transfer inhibiting capacity, PTIC). In seven animals the rise in level of circulating antibody occurred immediately before or in synchrony with the development of EAMG and frequently at such times serum complement rose irregularly. The PTIC was elevated during appearance of EAMG. In some animals a rise in complement fixing antibody level occurred without appearance of EAMG; in two others EAMG appeared without significant rise in antibody level. The data indicate that development of EAMG is associated with the production of antibodies which are capable of depressing neuromuscular transmission by reducing the sensitivity of the postjunctional membranes to acetylcholine. This depression can be potentiated by serum complement. Some but not all of the antibodies produced appear to fix complement when combined with Torpedo AChR. Evidence indicating possible existence of a presynaptic contribution to the development of EAMG is given.

Myasthenia in frogs immunized against cholinergic-receptor protein.

Frogs immunized with cholinergic-receptor protein developed myasthenia in 116--175 days. The muscular weakness was overcome by subcutaneous administration of 20 microgram of neostigmine. Electromyograms showed a decline in action potential amplitude during a 2-Hz train. Nerve stimulation evoked subthreshold end-plate potentials (EPPs) averaging 10.4 +/- 7.4 mV, but at many junctions no EPP was obtained. Miniature EPP amplitude had a modal value of 0.15 mV compared with 0.35 mV for the controls. The corresponding means were 0.24 +/- 0.23 mV and 0.48 +/- 0.23 mV. Microperfusion with edrophonium (5 mg/l) increased the amplitude of EPPs and miniature end-plate potentials (MEPPS). Postjunctional response tested with 20 muM carbamylcholine was 56% of control. Postjunctional response by carbamylcholine iontophoresis gave 19 +/- 22 mV/nC compared with 76 +/- 50 mV/nC for the controls. The data indicate that the neuromuscular transmission deficits in receptor-immunized frogs are mainly postsynaptic in origin, but there may be additional presynaptic contributions. This amphibian model of myasthenia gravis offers many opportunities and advantages in the study of receptor-immunized animals.

Reversible depletion of synaptic vesicles induced by application of high external potassium to the frog neuromuscular junction.

1. Reversible depletion of synaptic vesicles from frog cutaneous pectoris neuromuscular junctions was studied by application of a Ringer solution containing 115 mM-K propionate.2. During the release of transmitter, the synaptic vesicle membrane is added to the axolemmal membrane. Under the conditions of high K(+)-induced release, the synaptic vesicle membrane accumulates as folds formed in the region of the axolemmal membrane between the active zones. In depleted terminals, large vesicular structures appear and the evidence shows that some of them (possibly all) are formed as axolemmal infoldings. During formation of such infoldings the active zones remain fixed in position with respect to the post-junctional membrane.3. During recovery in normal Ringer solution, which followed 30 min depolarization in high K(+) Ringer solution, spontaneous m.e.p.p.s were detected as early as 9 min after the start of the recovery period and the average time for their reappearance was 17 min.4. At the end of a 20 min recovery period which followed K(+) depolarization, small accumulations of synaptic vesicles were again found within the terminal close to the active zones. At this time coated vesicles and coated pits were seen associated with the prejunctional axolemma and its infoldings. It appears that synaptic vesicles are re-formed directly from these coated vesicles.5. After 60 min recovery from K(+) depolarization, at which time stimulation of the motor nerve induced a muscle twitch, the structure of the terminals closely resembled that of control preparations.6. The entire synaptic vesicle recycling process can take place in the absence of the neurone soma.

Dipping cone to correct optical distortions at liquid surfaces.

A dipping cone attachment for use with a long-working-distance objective is described. The device eliminates degradation of the microscope image which occurs when microelectrodes are applied to single muscle fibers in vitro.

Pre- and post-junctional effects of 1-fluoro-2,4-dinitrobenzene at the frog neuromuscular junction.

1. FDNB increased by 60-90% the depolarization of the end-plate produced by applied carbachol in frog sciatic nerve-sartorius muscle preparations.2. In partially curarized preparations, FDNB (0.4 mM) increased the amplitude of the end-plate potential by a factor of 1.8.3. The quantal content of end-plate potentials was increased by FDNB (2 mM) as determined by the method of failures.4. After approximately 25-35 min, neuromuscular transmission was blocked by 0.4 mM-FDNB, as evidenced by abolition of neurally elicited end-plate potentials. At this stage miniature end-plate potentials could still be recorded, which indicates that the neuromuscular block was presynaptic.5. FDNB (0.4 mM) increased miniature end-plate potential frequency several hundred-fold when the Ringer solution contained normal calcium concentration (1.8 mM) or 0.45 mM calcium and 5.4 mM magnesium.6. During the first 60 min of exposure to 0.4 mM-FDNB there was a slight drop (4-6 mV) in resting potentials of muscle fibres. During this period directly initiated action potentials showed a marked decrease in the rate of repolarization and a small decrease in the amplitude and rate of rise.7. Using the technique of point voltage clamping in tetrodotoxin-treated muscles, it has been found that FDNB almost completely abolished the active increase in g(K) during stepwise depolarization of the nonjunctional muscle fibre membrane from -90 to 0 mV. The passive outward leakage current appeared unaffected by FDNB.

Factors in the inactivation of postjunctional membrane receptors of frog skeletal muscle.

Several factors which influence the rate of inactivation of muscle postjunctional membrane (PJM) receptors during the sustained application of carbamylcholine (CARB) have been studied by two methods. The rate of inactivation was increased by elevating the tonicity of the bathing medium, by increasing the CARB concentration, by raising the calcium ion concentration, and by substituting SO(4) (=) for Cl(-) ions in the extracellular fluid. The relative effectiveness of calcium and other divalent cations in receptor inactivation was compared. In the absence of calcium, other divalent cations such as magnesium, strontium, or manganese were not efficient substitutes for calcium. In the presence of calcium, the addition of strontium or manganese ions accelerated the rate of receptor inactivation, but the addition of magnesium (up to 12 mM) inhibited this process. The inactivation of the membrane receptors in denervated muscle fibers was found to be similar to that in innervated muscle fibers. Various factors in PJM receptor inactivation are discussed. It is suggested that PJM receptor inactivation is influenced by the binding of calcium ions to sites on the internal surface of the PJM.