C. G. Caratsch

Sequencing and synthesis of pardaxin, a polypeptide from the Red Sea Moses sole with ionophore activity

Pardaxin, an amphipathic polypeptide secreted by the Red Sea flatfish Pardachirus marmoratus whose sequence is NH2‐G‐F‐F‐A‐L‐I‐P‐K‐I‐I‐S‐S‐P‐L‐F‐K‐T‐L‐L‐S‐A‐V‐G‐S‐A‐L‐S‐S‐S‐G‐G‐Q‐E, was synthesized by the solid‐phase method. The structure was verified by sequencing. The synthetic polypeptide changed the resistance of lipid bilayers by forming pores. At 10−7−10−8 M, the synthetic pardaxin increased the frequency of the spontaneous release of quanta of acetylcholine at the neuromuscular junction by up to 100‐fold, resembling the native product. Synthetic pardaxin seems to be a suitable tool for investigating the molecular structures underlying channel selectivity.

Effects of phorbol ester on spontaneous transmitter release at frog neuromuscular junction.

Spontaneous transmitter release was studied at frog neuromuscular junctions exposed to the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), a specific activator of protein kinase C (PrkC). TPA at concentrations between 10−7 and 10−6 M induced a dose-dependent increase in miniature end-plate potential frequency. This frequency increase was enhanced by raising [Ca]0, diminished by lowering the temperature of the bath and virtually abolished in Ca2+-free Ringer. The TPA effect was only poorly reversible after washing. TPA was ineffective in increasing spontaneous release of transmitter at phosphatidylcholine-pretreated neuromuscular junctions. It is suggested that PrkC might play a role in neuromuscular transmission processes.

Presynaptic effects of the pardaxins, polypeptides isolated from the gland secretion of the flatfish Pardachirus marmoratus.

The effects of the two toxic proteins Pardaxin I and II isolated from the gland secretion of the flatfish Pardachirus marmoratus on frog neuromuscular transmission have been investigated and compared to those of the gland secretion. Pardaxin I and II showed pre- but not postsynaptic neurotoxic effects. They increased the frequency of the spontaneous release of transmitter quanta in a dose-dependent and temperature-influenced way up to more than 100 times control values. At the same time the quantal content of the evoked end-plate potentials was greatly elevated. Pardaxin I was about 5 times more effective than Pardaxin II, and both were roughly in the same range of efficacy as the original gland secretion (w/v). The glycosteroids isolated from the same gland secretion were relatively ineffective in promoting neurotransmitter release; however, at high doses they had postsynaptic effects, as shown by a diminution of the amplitude of the evoked end-plate potentials. They did not reinforce the effect of the Pardaxins. At higher doses both the Pardaxins and the gland secretion induced depolarization of postsynaptic membranes, muscle cell contractions which could not be blocked by (+)-tubocurarine or by tetrodotoxin, and eventually also physical disruption of muscle cells. No effects on nerve conductance were observed. Pore-forming activity of the Pardaxins has already been demonstrated. It is suggested that their presynaptic effects are a result of a possible affinity to the nerve terminals, of their hydrophobicity and mainly of this pore-forming activity. These toxins might be valuable tools in neuroscience research.

Influence of protein kinase C-stimulation by a phorbol ester on neurotransmitter release at frog end-plates

Summary1. The effect of the phorbol ester 12-O-tetradecanoylphorpbol-13-acetate (TPA) on the stimulation-evoked neurotransmitter release has been investigated by measuring the quantal content (m) of end-plate potentials at frog neuromuscular junctions (Rana temporaria, M. sartorius). 2. After addition of TPA (0.1 up to 1 μmol/1) to the Ringer solution the m-values increased in a concentration-dependent manner up to more than 3 times the control values. 3. Inhibition of the activity of the protein kinase C through the inhibitor 1(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) blocked this effect of TPA. 4. The TPA effect was much more conspicuous when the m-value was reduced by raising the extracellular Mg2+ concentration. Between the control m-values and the n-fold increase in the m-value enhanced by TPA a hyperbolic relation was observed. 5. It is concluded that protein kinase C stimulation affects predominantly the spontaneous release of neurotransmitter at the frog neuromuscular junction and only very poorly the stimulation-evoked one.

Postsynaptic effects of the phorbol ester TPA on frog end-plates

The effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a specific activator of protein kinase C (PKc), were examined on the frog neuromuscular junction. The depolarization elicited by iontophoretically applied acetylcholine (ACh) was reversibly decreased by 20–60% when muscle fibres were exposed to 1–5×10−7 M TPA. Liposome-delivered phosphatidylcholine (100 μg/ml) prevented this effect. A similar decrease in ACh-sensitivity was produced by diacylglycerol (diolein), a physiological activator of PKc, but in this case the decrease was only partially reversible. In TPA-Ringer, (1) the peak size of miniature end-plate potentials exhibited a small decrease; (2) miniature end-plate currents were reduced in size and their decay time constant became longer and relatively independent of membrane potential. The possibility that these TPA-induced actions are mediated by activation of PKc is discussed.

Functional regulation of nicotinic acetylcholine receptor channels in muscle.

Increasing evidence indicates that the function of membrane ligand-operated ion channels may be changed in response to hormones, neurotransmitters, and peptides. An important mechanism for signal transduction has been identified in protein phosphorylation. This event is the final step of a cascade process consisting of:

Interleukin-2 lengthens extrajunctional acetylcholine receptor channel open time in mammalian muscle cells

The effect of interleukin-2 (rIL-2) on nicotinic acetylcholine receptors (nAChR) was examined on cultured muscle fibres isolated from the flexor digitorum brevis muscle (FDB) of the rat and on aneural mouse cultured C2 myotubes. Intracellular measurement of the sensitivity to iontophoretically applied ACh demonstrated that the sensitivity of the extrajunctional nAChRs in cultured fibres showed a transient increase after application of rIL-2 (2,000–3,000 units/ml). Cell-attached patch-clamp experiments on the same fibres proved that rIL-2 (2,000 units/ml) induces a significant increase in the mean open time of the extrajunctional nAChR channel. The other channel parameters were not significantly modified. The same applied also to aneural mouse patch-clamped C2 myotubes exposed to rIL-2 (2,000 units/ml). In freshly dissociated fibres no effects on nAChR channels were observed following rIL-2 application. 125I-rIL-2 binding experiments on either 7-day cultured or freshly dissociated adult muscle fibres showed that a specific binding with a Kd of 2.07±0.4 nM develops in cultured fibres but fails to occur immediately after dissociation. It is concluded that rIL-2 modulates the duration of extrajunctional nAChR channels in both myotubes and adult muscle cells, and that this effect is probably due to the activation of a second messenger system.

Effects of obidoxime chloride on native and sarin-poisoned frog neuromuscular junctions

Abstract1.The effect of the oxime reactivator obidoxime chloride (obidoxime) on single frog neuromuscular junctions has been studied in order to clarify its action on the acetylcholine receptor (AChR) and on the acetylcholine esterase (AChE), both before and after blocking its enzymatic activity with the organophosphorus compound sarin.2.Experiments with iontophoretic application of obidoxime to end-plates demonstrated that it has a weak direct depolarizing effect. Furtheron, the drug is shown to possess a potentiating effect on the ACh-induced depolarization. After the AChE activity had been inhibited with sarin, obidoxime on the contrary decreases the depolarization induced by ACh. Both effects are fully reversible.3.It is concluded that obidoxime acts as an inhibitor of the AChE and as a partial antagonist of the AChR. The antagonistic effect on the receptor is usually masked by the predominating anticholinesterase effect.4.The effect of obidoxime on miniature end-plate potentials in long-time experiments on sarin-poisoned muscles, showed only weak signs of recovery from the action of the AChE inhibitor. Only focally higher concentration of the drug produced a more marked but short term recovery of the mepps, which is, however, supposed to be dependent on the AChR antagonism.5.It is still unclear how much of the varying therapeutic usefulness of obidoxime in clinical cases is due to its AChE reactivation and how much to the antagonistic effect on the AChR.

Regulation of acetylcholine receptor function by the phorbol ester TPA in rat skeletal muscle

Summary(1) The effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a specific activator of the protein kinase C (PrkC), on the function of junctional nicotinic acetylcholine receptors (nAChR) was examined on muscle fibres isolated from the M. flexor digitorum brevis of the rat. (2) In the presence of TPA the sensitivity of the whole endplates to iontophoretically applied ACh exhibited multiphasic oscillations: an early decrease followed by a delayed increase and, at the end again, a decrease to below pretreatment levels. This effect was more pronounced as the TPA concentration was increased in the range of 0.1–1 μM and was blocked by the PrkC-inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). (3) TPA (0.1–0.5 μM) shortly applied to patch-clamped fibres caused a slight decrease in nAChR-channel slope conductance without affecting the mean lifetime. In a patch the opening frequency increased over time, after an initial decrease. (4) It ist concluded that specific activation of the PrkC may be of regulatory significance on nAChR function.

Quantitative correlation between complete block of nicotinic acetylcholine receptors and saturation of the motor endplate with 14C-toxiferine.

Summary1.To study the quantitative correlation between the stabilizing effect of toxiferine on the postsynaptic membrane and the number of drug binding sites at the motor endplate, experiments were performed on isolated mouse hemidiaphragms using combined electrophysiological and autoradiographic techniques.2.The membrane stabilizing effect of 14C-toxiferine was investigated over a wide range of concentrations, in order to obtain not only muscle paralysis but the complete abolition of the endplate response to nerve stimulation.3.Motor endplate activity was recorded intracellularly. For each concentration of the drug the percentage of endplates reacting to nerve stimulation with action potentials (a.p.), or with subliminal endplate potentials (e.p.p.), or being completely blocked, was determined. The mean amplitude of the e.p.p.s was also calculated.4.Reduction of the e.p.p. to an undetectable level required a concentration of 14C-toxiferine about 3 times greater than that which induced complete paralysis.5.Correlation of these data with autoradiographic measurements demonstrated that decreasing e.p.p. amplitude corresponded to increasing values for the number of drug binding sites per endplate. Saturation was reached by that concentration which completely blocked the postsynaptic sensitivity to ACh.6.It is concluded that at this concentration all the specific drug binding sites are occupied.