Koh-Ichi Enomoto

Effects of synthetic ω-conotoxin, a new type Ca2+ antagonist, on frog and mouse neuromuscular transmission

A new type Ca2+ antagonist, synthetic omega-conotoxin (omega-CgTX) decreased the peak height of the endplate potential (EPP) in frog muscle but had no effect on the mouse neuromuscular junction. The reduction of endplate potential in frogs was due to a decrease in transmitter release, since the mean quantal content estimated by variance of EPPs (m) and from the peak heights of EPPs and miniature EPPs (m1) was reduced by omega-CgTX, but the postsynaptic sensitivity to ACh was unaltered. The decrease of mean quantal content caused by omega-CgTX was reversed by 4-aminopyridine, guanidine and Bay K 8644. Also, the effect of omega-CgTX was weakened in the presence of 10 mM Ca2+ or 12 mM Mg2+. Statistical analysis revealed that omega-CgTX decreased the number of quanta available (n) whereas the probability of release (p) remained unaffected.

Effects of memantine on the frog neuromuscular junction

The effects of memantine, an adamantane derivative, on neuromuscular transmission in the frog sartorius muscle preparation were studied by measuring the endplate current (EPC) by the voltage clamp method. Memantine (0.5-50 microM) reduced the peak amplitude and shortened the duration of the EPC, and the membrane voltage-peak EPC relationship became non-linear. Since it decreased the quantal height of the EPC without affecting the mean quantal content, the effects were considered to be mainly postsynaptic. Noise analysis revealed that memantine decreased the mean lifetime of the ACh-activated channel. In addition, lineweaver-Burk analysis of the response to ionophoretically applied carbamylcholine showed that memantine acted essentially as a linear mixed-type inhibitor. Thus memantine seems to block neuromuscular transmission by reacting with the ACh receptor-ion channel complex in both the open and closed states.

Single calcium-activated potassium channel in cultured mammary epithelial cells

The properties of Ca2+-activated K+ channels in mouse mammary epithelial cells in primary culture were studied by the patch-clamp technique. In cell-attached patches, spontaneous channel openings were sometimes observed; the slope conductance of the currents was about served; the slope conductance of the currents was about 12 pS at negative membrane potentials with a physiological solution (152 mM Na+, 5.4 mM K+) in the pipette. External application of A23187, a calcium ionophore, activated this channel. In excised inside-out patches, the channel was activated by increasing the internal Ca2+ concentration (10−7 to 10−6 M). No voltage dependence of the channel activity was observed. Internal Na+ blocked the outward K+ current in a voltage dependent manner and this block led to the non-linear I–V relationship at positive membrane potentials. The channel was blocked by internal Ba2+ (0.1 mM) and tetracthylammonium (TEA+, 20–50 mM). Ba2+ reduced the open probability but not the single channel conductance, whereas TEA+ reduced the single channel conductance. The single channel conductance of this channel, measured from the inward current with a high-K+ solution (150 mM K+) in the pipette, was large (about 40 pS), and showed inward rectification. These results suggest that this channel is different from the usual small conductance Ca2+-activated K+ channels observed in many other cells.

Oscillating activity of a calcium-activated K+ channel in normal and cancerous mammary cells in culture

SummaryCalcium-activated potassium channels were the channels most frequently observed in primary cultured normal mammary cell and in the established mammary tumor cell, MMT060562. In both cells, single-channel and whole-cell clamp recordings sometimes showed slow oscillations of the Ca2+-gated K+ current. The characteristics of the Ca2+-activated K+ channels in normal and cancerous mammary cells were quite similar. The slope conductances changed from 8 to 70 pS depending on the mode of recording and the ionic composition in the patch electrode. The open probability of this channel increased between 0.1 to 1 μm of the intracellular Ca2+, but it was independent of the membrane potential.Charybdotoxin reduced the activity of the Ca2+-activated K+ channel and the oscillation of the membrane current, but apamin had no apparent effect. The application of tetraethylammonium (TEA) from outside and BaCl2 from inside of the cell diminished the activity of the channel. The properties of this channel were different from those of both the large conductance (BK or MAXI K) and small conductance (SK) type Ca2+-activated K+ channels.

Mechanically induced electrical responses in murine mammary epithelial cells in primary culture

In mouse mammary epithelial cells in primary culture, mechanical stimulation of a cell induced in other cells within the same colony a short depolarization of less than 15 m V with a duration of 1–8 s and a subsequent, prominent hyperpolarization of 6 m V lasting 10–40 s. Epidermal growth factor induces a spontaneous hyperpolarizing response in cultured mammary cells, and in cells treated with EGF mechanical stimulation produced a greater hyperpolarization, while the amplitude of the depolarizing response was not affected. The amplitude of the mechanically induced hyperpolarization was markedly reduced by quinine and tetraethylammonium, blockers of the Ca2+‐dependent K+ channel. The results suggest that the Ca2+‐dependent K+ channel was involved in the hyperpolarization.

Real-time imaging of intracellular calcium change with simultaneous single channel recording in mammary epithelial cells

A method of continuous image subtraction of fura-2 fluorescence made it possible to observe real-time (video rate) changes in intracellular calcium (Cai). This simple method is very useful for simultaneous measurements in electrophysiology with a system containing cells of different states. A method for synchronous recording of Cai change and single channel activity is also described. In cultured mammary epithelial cells, these methods revealed a propagating Cai signal induced by mechanical stimulation and spontaneous Cai oscillation with synchronous activation of calcium-activated potassium channels.

Involvement of the Ca2+-dependent K+ channel activity in the hyperpolarizing response induced by epidermal growth factor in mammary epithelial cells

Epidermal growth factor (EGF) induces a hyperpolarizing response of 5–20 mV amplitude in mouse mammary epithelial cells in culture. The amplitude of the hyperpolarizing response was reduced by more than 60% within several minutes after addition of blockers of voltage and/or Ca2+‐dependent K+ channels such as tetraethylammonium (7 mM) or quinine (0.29 mM). Both nifedipine (0.15 mM), a blocker of the Ca2+ channel, and ruthenium red (2 mM), an inhibitor of the Ca2+‐binding site, also reduced the amplitude of the hyperpolarizing response by more than 60%. The Ca2+ ionophore, A23187 (3.8 μM), induced a large hyperpolarization, which was 25–40 mV and lasted about 3 min. These data suggest that activity of the Ca2+‐dependent K+ channel was involved in the EGF‐induced hyperpolarizing response of the mammary epithelial cells.

Presynaptic effects of 4-aminopyridine and streptomycin on the neuromuscular junction

Studies were done to assess the effects of 4-aminopyridine (4AP) and streptomycin (SM) on transmitter release parameters and extracellularly recorded presynaptic action potential. The application of 5 micrometer 4AP resulted in a marked increase in the mean quantal content (m1) associated with an increase in the total number of the store of available quanta (n) but had no effect on the probability of release (p) and the fractional release (P). Focal recording showed that 50 micrometer 4AP modified the shape of the presynaptic action potential from a triphasic configuration to a diphasic one. In contrast, prolongation of the muscle action potential was found only at higher concentrations (greater than 1 mM) of of 4AP. Thus, the increase in n with 4 AP was associated with prolongation of the presynaptic action potential evoked by blocking the K current. SM (172 micrometer mM) had no effect on p and P. Reduction of n by SM was completely reversed by 4AP. As the presynaptic action potential change induced by 4AP was not antagonised by SM, it may be that the decrease of n by SM followed a modification of the voltage-dependent Ca channel.

A microcomputer system for on-line analysis of the end-plate potential data.

Abstract A low cost signal processor using a microcomputer and a digital memory for on-line analysis of transmitter release parameters has been produced. Program for analysis has been written in BASIC and ASSEMBLER.

Kinetic analyses of transmitter release in neuromuscular transmission.

A majority of transmitter quanta in the nerve terminal is in a large pool of store (S), which can be utilized for release only after transformation into activated quanta (n) via two intermediate states called available quanta (A) and releasable quanta (N). Mobilization is a collective term applicable for aggregates of S----A, A----N and N----n conversions. In the present article, various electrophysiological procedures for kinetic analyses of the transmitter release in neuromuscular transmission were discussed to elucidate this not well understood process of mobilization. Especially, frequency augmentation, tetanic rundown and depression, and recovery from depression experiments were proposed to be very useful tools in identifying the drug effect on the mobilization process. Since d-tubocurarine, 2-(4-phenylpiperidino)cyclohexanol (AH5183) and Ba ions satisfied the above three criteria of drug action on mobilization, these were concluded to affect the mobilization of transmitter in neuromuscular transmission.