Block of Quantal End-Plate Currents of Mouse Muscle by Physostigmine and Procaine

of quantal end-plate currents of mouse muscle by physostigmine and procaine. Quantal endplate currents (qEPCs) were recorded from hemidiaphragms of mice by means of a macro-patch-clamp electrode. Excitation was blocked with tetrodotoxin, and quantal release was elicited by depolarizing pulses through the electrode. Physostigmine (Phys) or procaine (Proc) was applied to the recording site by perfusion of the electrode tip. Low concentrations of Phys increased the amplitude and prolonged the decay time constants of qEPCs from ∼3 to ∼10 ms, due to block of acetylcholine-esterase. With 20 μM to 2 mM Phys or Proc, the decay of qEPCs became biphasic, an initial short time constant τs decreasing to <1 ms with 1 mM Phys and to ∼0.3 ms with 1 mM Proc. The long second time constant of the decay, τl, reached values of ≤100 ms with these blocker concentrations. The blocking effects of Phys and Proc on the qEPC are due to binding to the open channel conformation. A method is described to extract the rate constants of binding ( b p) from the sums 1/τs + 1/τl, and the rates of unbinding ( b −p) from τ0 · τs −1 · τl −1 (τ0 is the decay time constant of the control EPC). For Phys and Proc b p of 1.3 and 5 · 106M−1 s−1 and b −p of 176 and 350 s−1, respectively, were found. Using these rate constants and a reaction scheme for the nicotinic receptor together with the respective rate constants determined before, we could model the experimental results satisfactorily.

Ca(2+)-induced Ca2+ release amplifies the Ca2+ response elicited by inositol trisphosphate in macrophages.

We have studied the rise in intracellular calcium concentration ([Ca2+]i) elicited in macrophages stimulated by platelet-activating factor (PAF) by using fura-2 measurements in individual cells. The [Ca2+]i increase begins with a massive and rapid release of Ca2+ from intracellular stores. We have examined the mechanism of this Ca2+ release, which has been generally assumed to be triggered by inositol trisphosphate (IP3). First, we confirmed that IP3 plays an important role in the initiation of the PAF-induced [Ca2+]i rise. The arguments are 1) an increase in IP3 concentration is observed after PAF stimulation; 2) injection of IP3 mimics the response to PAF; and 3) after introduction of heparin in the cell with a patch-clamp electrode, the PAF response is abolished. Second, we investigated the possibility of an involvement of Ca(2+)-induced Ca2+ release (CICR) in the development of the Ca2+ response. Ionomycin was found to elicit a massive Ca2+ response that was inhibited by ruthenium red or octanol and potentiated by caffeine. The PAF response was also inhibited by ruthenium red or octanol and potentiated by caffeine, suggesting that CICR plays a physiological role in these cells. Because our results indicate that in this preparation IP3 production is not sensitive to [Ca2+]i, CICR appears as a primary mechanism of positive feedback in the Ca2+ response. Taken together, the results suggest that the response to PAF involves an IP3-induced [Ca2+]i rise followed by CICR.

Reversal potential of GABA-induced currents in rod bipolar cells of the rat retina

GABA-induced whole-cell currents were measured in rod bipolar cells dissociated from the adult rat retina. The patch-clamp electrode contained nystatin, which made the cell membrane electrically permeable without rupture, thus retarding the rate of diffusion of Cl– ions from the patch pipette to the cell interior. The reversal potential of the GABA-induced currents was around –70 mV at an extracellular Cl-–concentration of 149 mM. We conclude that GABA generates hyperpolarizing responses in rod bipolar cells of the rat retina.