Yong I. Kim

Inhibition of calcium currents and exocytosis by Lambert‐Eaton syndrome antibodies in human lung cancer cells.

1. Human small‐cell lung cancer (SCLC) cells are believed to express the antigens responsible for the production of pathological antibodies in the Lambert‐Eaton syndrome (LES), a Ca2+ channel disorder in which quantal transmitter release from the motor nerve terminal is impaired. Whole‐cell patch‐clamp techniques were used to study the voltage‐dependent Ca2+ channels expressed by H146 SCLC cells and the effects of LES antibodies on these channels. The types of Ca2+ channels were determined using biophysical properties and pharmacological sensitivity to several antagonists. 2. Whole‐cell Ca2+ currents (ICa) in SCLC cells are sensitive to the dihydropyridine (DHP) nicardipine, omega‐conotoxin GVIA (omega‐CgTX GVIA) and omega‐agatoxin IVA (omega‐AgTX IVA). Nicardipine at 100 nM and 10 microM reduced ICa by 35 and 45% (n = 38 cells), respectively, while omega‐CgTX GVIA (1 microM) inhibited ICa by 32% (n = 31). Application of omega‐AgTX IVA at 50 and 100 nM to the cancer cells decreased ICa by 41 and 42%, respectively (n = 22). 3. Measurement of cell membrane capacitance (Cm) revealed that Ca(2+)‐dependent exocytosis underlies the secretory activity of SCLC cells. Exocytosis, when induced by step depolarizing pulses and measured by increases in Cm, was markedly inhibited by nicardipine (10 microM) and omega‐AgTX IVA (100 nM). In contrast, omega‐CgTX GVIA (1 microM) was not as effective in altering increases in Cm. 4. From negative (‐80 mV) and depolarized (‐40 mV) holding potentials, both peak and plateau ICa were inhibited by the presence of LES antibodies (1 mg ml‐1 IgG). LES serum also reduced depolarization‐induced increases in Cm by 48% (n = 15). 5. To determine whether the LES antibodies are downregulating a specific type(s) of Ca2+ channel, nicardipine (10 microM), omega‐CgTX GVIA (1 microM) or omega‐AgTX IVA (100 nM) was applied to tumour cells that had been previously exposed to LES serum for 24 h. The most pronounced change was that omega‐AgTX IVA was 38‐84% less effective at reducing ICa after the IgG treatment. The effectiveness of nicardipine was diminished by 18% after incubation with the LES antibodies, whereas the omega‐CgTX GVIA was seen to be more effective. These results suggest that LES IgG downregulates P‐type Ca2+ channels and, possibly, to a lesser extent L‐type channels. 6. In view of recent evidence that P‐type Ca2+ channels mediate cholinergic transmitter release at the mammalian neuromuscular junction (NMJ), the expression of P‐type Ca2+ channels in the SCLC cells and the reactivity of LES IgG with these channels support the hypothesis that P‐type Ca2+ channels in these cancer cells may trigger the autoantibody production in this disorder. The antibodies so produced are implicated in the functional impairment of the Ca2+ channels characteristic of LES.

Eaton-Lambert syndrome: A clinical and electrophysiological study of a patient treated with 4-aminopyridine

In a patient with the Eaton-Lambert syndrome, 4-aminopyridine produced temporary improvement of clinical and electromyographic abnormalities. Application of the drug in vitro to intercostal muscle from the patient produced an increase in the evoked release of neurotransmitter from intramuscular nerves.

Fluorescence emission spectral shift measurements of membrane potential in single cells.

Previous measurements of transmembrane potential using the electrochromic probe di-8-ANEPPS have used the excitation spectral shift response by alternating excitation between two wavelengths centered at voltage-sensitive portions of the excitation spectrum and recording at a single wavelength near the peak of the emission spectrum. Recently, the emission spectral shift associated with the change in transmembrane potential has been used for continuous membrane potential monitoring. To characterize this form of the electrochromic response from di-8-ANEPPS, we have obtained fluorescence signals from single cells in response to step changes in transmembrane potentials set with a patch electrode, using single wavelength excitation near the peak of the dye absorption spectrum. Fluorescence changes at two wavelengths near voltage-sensitive portions of the emission spectrum and shifts in the complete emission spectrum were determined for emission from plasma membrane and internal membrane. We found that the fluorescence ratio from either dual-wavelength recordings, or from opposite sides of the emission spectrum, varied linearly with the amplitude of the transmembrane potential step between -80 and +60 mV. Voltage dependence of difference spectra exhibit a crossover point near the peak of the emission spectra with approximately equal gain and loss of fluorescence intensity on each side of the spectrum and equal response amplitude for depolarization and hyperpolarization. These results are consistent with an electrochromic mechanism of action and demonstrate how the emission spectral shift response can be used to measure the transmembrane potential in single cells.

INTERCOSTAL MUSCLE BIOPSY STUDIES IN MYASTHENIA GRAVIS: CLINICAL CORRELATIONS AND THE DIRECT EFFECTS OF DRUGS AND MYASTHENIC SERUM *

We have found a wide range of mean MEPP amplitude in intercostal muscle biopsies from 43 patients with MG, including several values in the normal range. There was no correlation between MEPP amplitude and the severity of clinical disease as assessed by manual muscle testing or by single-fiber EMG measurements of jitter in arm muscles. Through most of these patients were in a state of clinical remission or marked improvement after treatment with prednisone, we could not attribute the difference between our results and those of others to this factor alone. The application of morphine, meperidine and aminoglycoside antibiotics to intercostal muscle in vitro confirms effects previously demonstrated in rat muscle: (1) At equal therapeutic concentrations, meperidine has greater neuromuscular blocking effects than does morphine, but neither has significant effects at concentrations achieved in the serum clinically. (2) Tobramycin, netilmicin and neomycin have varying severity and sites of action, but their effects are the same in human myasthenic muscle as in normal rat muscle. Bath application of serum from myasthenic patients produces an acute, reversible worsening of neuromuscular blockade in myasthenic muscle. Electrophysiologic measurements in intercostal biopsies from patients with MG can provide information about the basic abnormality of neuromuscular transmission in this disease and can confirm the relevance of studies made in animal muscle.

Maitotoxin activates quantal transmitter release at the neuromuscular junction: evidence for elevated intraterminal Ca2+ in the motor nerve terminal

Maitotoxin (MTX), applied in vitro to the mouse neuromuscular junction, briskly activates the spontaneous release of acetylcholine quanta, manifest as miniature end-plate potentials (MEPPs). This effect requires external Ca2+ and is accompanied by a steady post-junctional depolarization. After the peak activation of the spontaneous release process, the quantal discharge gradually declines with eventual abolishment of MEPPs. In contrast to the striking increase in MEPP frequency, the quantum content of the nerve-evoked end-plate potentials (EPPs) is increased only moderately by MTX. These effects are attributable to the ability of the toxin to elevate the level of free intracellular Ca2+ in the motor nerve terminal. With further characterization of its presynaptic site of action, maitotoxin may become a useful tool in studying synaptic physiology.

Radial decline of the extracellular action potential

A modified line source model presented earlier has been used to study the decline of the extracellular single muscle fibre action potential. The muscle tissue is modelled as a low-pass filter. The transfer function of the filter declines more slowly than a first order low-pass filter at low frequencies, but much faster at high frequencies. The cutoff frequency of the filter increases when the anisotropy of the muscle decreases. It also increases proportionally with the propagation velocity of the action potential. The decline of different frequency components obtained from the modified line source volume conductor and a filter model derived from experimental measurements are compared and their differences explained. The modified line source model was found to be identical to the volume conductor model in terms of results and at the same time conceptually simple for applications.

Short-term effects of prednisolone on neuromuscular transmission in normal rats and those with experimental autoimmune myasthenia gravis.

Electrophysiological investigations of the effects of bath-applied prednisolone at the neuromuscular junction were performed in muscles from normal rats and rats with experimental autoimmune myasthenia gravis (EAMG). In muscles from both groups, prednisolone reversible and significantly depressed the amplitudes of minature end-plate potentials (MEPPs), end-plate potentials (EPPs) and indirectly elicited action potentials (APs) without affecting resting membrane potentials. Prednisolone also caused a significant reduction in EPP rise time to peak and half-decay time while markedly increasing MEPP frequency and AP rise time to peak and duration. These effects were shown to be dose-dependent. The percentage decrease in amplitude after prednisolone perfusion was similar for EPPs and MEPPs, indicating that the depressive effect of prednisolone at the junction is postsynaptic. In all of the parameters studied, the percentage effect of prednisolone was the same in EAMG and normal preparations. No stimulus-linked repetitive EPPs or APs were observed after prednisolone. It is concluded that prednisolone has a depressive effect on neuromuscular transmission, but that this occurs only at high concentrations of the drug which are not achieved during the treatment of myasthenia gravis.

Dopamine enhances a voltage-dependent transient K+ current in the MMQ cell, a clonal pituitary line expressing functional D2 dopamine receptors

The influence of dopamine on voltage-dependent K+ current (IK) was studied in cultured MMQ cells using the whole-cell patch-clamp technique. IK in nearly all MMQ cells revealed a transient outward current component and inactivated during maintained depolarization lasting 60 ms. The transient component was inhibited by prepulse potentials more positive than -40 mV or by addition of 4 mM 4-aminopyridine to the bathing solution and was insensitive to the external Ca2+ concentration. Thus, this transient K+ current resembled the A-current (IA) found in other cells. Dopamine at 1 microM increased by 50% (P less than 0.001) the peak of IK evoked by a test potential to +80 mV and the response was prevented by pretreatment with 100 nM haloperidol, a D2 receptor antagonist. These data suggest that MMQ clonal pituitary cells possess a voltage-gated K+ A-current and that this current can be modulated by dopamine via D2 receptors.

A computer system for real-time data acquisition and analysis of biopotentials and quantal content at the neuromuscular junction.

A computerized data acquisition system for on-line analysis of the parameters of neuromuscular transmission is described. Both hardware usage and software methodologies are discussed with regard to sampling in real-time and analyzing miniature end-plate potentials (MEPPs), end-plate potentials (EPPs) and quantal content of the evoked transmitter release. Significant features of the program include: (1) automatic threshold determination for MEPP detection; (2) the use of a circular buffer to give pre-trigger information; (3) real-time noise spike rejection; (4) an automatic procedure for EPP failure detection; (5) rapid quantal content determinations by several methods as well as complete MEPP and EPP waveform analysis. The system has proven both accurate and reliable during more than two years of use. Advantages of the system over conventional methods include: (1) increased accuracy and efficiency in data analysis; (2) immediate availability of results; (3) conventional data storage; (4) flexibility to meet changing requirements.

Effects of Bay K 8644 on spontaneous and evoked transmitter release at the mouse neuromuscular junction

The dihydropyridine, Bay K 8644, was applied in vitro to mouse phrenic nerve-diaphragm muscle preparations. The drug increased both spontaneous and evoked release of acetylcholine from the motor nerve terminal in a concentration- and time-dependent manner. The rise in miniature endplate potential frequency, however, was the result of an increased intraterminal mobilization of free calcium, rather than well-established activation of voltage-dependent calcium channels. This view is supported by the following observations: (1) an increase in frequency was apparent in Ca2+-free medium; (2) Bay K 8644 is known to require a moderate depolarization to affect Ca2+ channels, but no membrane depolarization was detected; and (3) exposure to low Ca2+ and high Mg2+ medium did not diminish the effect on miniature endplate potential frequency. In a medium containing low Ca2+ and high Mg2+, Bay K 8644 increased quantal content of the evoked endplate potentials to a greater degree and with a faster time course than the frequency of miniature endplate potentials. This enhancement in evoked release did not appear to be caused solely by an increase in cytoplasmic Ca2+, but rather reflected at least in part the Bay K 8644-induced activation of voltage-gated Ca2+ channels, perhaps L-type, at the presynaptic nerve terminal. Thus, we propose that Bay K 8644 exerts dual effects on the motor nerve endings, characterized by a primary action on the presynaptic Ca2+ channels and a secondary action associated with the elevation of intracellular Ca2+ concentration.