Yoshimi Ikemoto

Enhancement by Propofol of the γ-aminobuiyric Acida Response in Dissociated Hippocampal Pyramidal Neurons of the Rat

BackgroundActivation of the γ-aminobutyric acidA (GABAA) receptor-ionophore complex has been reported as a possible molecular mechanism of the anesthetic action of propofol. Augmentation of GABA-induced inhibitory transmission has also been suggested as a mechanism. Because data describing this latter mechanism in mammalian neurons are few, we have examined the effects of propofol on the GABA response in central neurons of the rat. MethodsHippocampal pyramidal neurons were dissociated after enzyme treatment of the rat brain slices. The neurons were voltage-clamped with the whole cell configuration of the patch clamp technique. Neurotransmitters and drugs were applied using the “Y-tube” method, which exchanges the extracellular solutions around the neuron within 10–20 ms and makes it possible to obtain the peak response before desen-sitization develops. ResultsIn pyramidal neurons voltage-clamped at −60 mV, GABA induced an inward current. Propofol (10-6 M) augmented the current and shifted the concentration-response curve for GABA to the left without affecting the maximum response. A low concentration of the anesthetic (10-6 M) reduced the dissociation constant for GABA from 8.2 × 10-6 to 4.2 × 10-6 M without a significant effect on the Hill coefficient. Coappli-cation of propofol at a higher concentration (5 × 10-6 M) also shifted the GABA dose-response curve to the left, reducing the dissociation constant to 2.8 × 10-6 M. Potentiation by propofol was not associated with a change in the reversal potential for the GABA response and was not voltage-dependent. The inhibitory glycine response was not affected by propofol (10-6 M or 5 × 10-6 M). ConclusionsPropofol at clinically relevant concentrations enhances the inhibitory GABAA receptor-mediated response in mammalian central neurons. The enhancement may result in reduced excitability of the neuronal network and may, consequently, contribute to the anesthetic action of the agent.

Reduction of the Slow Inward Current of Isolated Rat Ventricular Cells by Thiamylal and Halothane

The barbiturates and halothane exert a negative inotropic effect on the myocardium. A reduction in the slow inward current, carried mainly by calcium ions, is an important factor for the underlying mechanism because the calcium current during the action potential provides the calcium ions for accompanying contraction, supplies Ca ions to the sarcoplasmic reticulum for subsequent contractions, and induces Ca release from the store site. It has been suggested that reduction in the slow inward current caused by anesthetics is indicated by depression of the slow action potential of the partially depolarized myocardium. In order to assess directly the effect of anesthetics on the slow inward current, we carried out voltage clamp experiments with single isolated rat ventricular cells obtained by an enzymatic dissociation method. Thiamylal (10‐4 mol·1‐1) and halothane (1%) decreased the slow inward current to 60 ± 5% (mean ± s. d., n = 8) and to 65 ± 10% (mean ± s. d., n = 8) of the control value, respectively, without changing the configuration of the current‐voltage curve. The results provide further evidence for anesthetic reduction of the slow inward current of the myocardium, and suggest that the negative inotropic effect is at least partly due to the reduction in that current.

Delayed activation of large-conductance Ca2+-activated K channels in hippocampal neurons of the rat.

We applied a fast concentration jump system to produce step changes in Ca2+ concentration [( Ca2+]i) on the cytoplasmic side of the inside-out membrane patch, excised from isolated rat hippocampal pyramidal neurons, and examined the time course of the activation phase of the large-conductance K channel (the BK channel; approximately 266 pS) after a step rise in [Ca2+]i. Diffusion of Ca2+ from the electrode tip to the cytoplasmic surface of the patch was estimated to be almost completed in 10 ms. After a step increase in [Ca2+]i from 0.04 to 3.2-1,000 microM, the activation of the K channel started after a clear latency of 280-18 ms and proceeded along a sigmoidal function. This was in sharp contrast with the rapid deactivation that began without delay and that was completed within 50 ms. The latency in activation was not accounted for by the binding of Ca2+ to EGTA in unstirred layers in the patch, since this binding was reported to be slow, taking up to seconds at physiological pH. Calmodulin (1 microM) did not affect the delay, the activation rate, or the steady-state current level. The calmodulin inhibitors W-7 and W-5 caused flickering of the single-channel current. These results indicate a delayed activation of the BK channel after a step rise in [Ca2+]i, suggesting that the BK current does not contribute to the repolarization of the action potential. Calmodulin is probably not involved in the activation process of the channel.

Blockade by local anaesthetics of the single Ca2+-activated K+ channel in rat hippocampal neurones

1 Effects of local anaesthetics on single Ca2+‐activated K+ channels were investigated using the inside‐out configuration of the patch‐clamp technique in single pyramidal neurones, which were freshly dissociated from rat hippocampus by use of proteolytic enzymes. 2 No significant effect was observed when 2 mm benzocaine was applied on either side of the membrane patch, or when 3 mm lignocaine or QX‐314 was applied to the external surface of the membrane. 3 Lignocaine 1 mm, applied to the internal surface, slightly reduced the amplitude of the single K+ channel current. When applied to the internal surface, QX‐314 reduced the amplitude of the K+ channel current, accompanied by an increase in noise in the open channel current, suggesting a fast flickering block. The blocking effect of QX‐314 on the outward current increased with depolarization, suggesting a binding site for the drug at an electrical distance of about 0.5 across the membrane field. 4 The open time histogram showed one exponential component and the closed time histogram showed at least two components. The mean open time of the outward current was increased when the amplitude was reduced by the drugs. 5 The ionized form of the local anaesthetics had a similar action on the Ca2+‐activated K+ channels to that on Na+ channels, that is, they enter into the channel from the cytoplasmic side to induce open channel block. The blocking kinetics, however, might be so fast that they were beyond the frequency response of our recording apparatus, thus the recorded current amplitude was decreased. In contrast the K+ channel was not accessible via hydrophobic pathways for the neutral form, which is also known to block the sodium channel.

Local anesthetics reduce the inhibitory neurotransmitter-induced current in dissociated hippocampal neurons of the rat

The effects of local anesthetics on amino acid-induced currents were examined using the whole-cell configuration of the patch clamp technique in dissociated hippocampal pyramidal neurons of the rat. Lidocaine (3 mM) decreased the glycine-induced Cl- current (Gly-ICl) more potently (to 46% of the control value) than the gamma-aminobutyric acid-induced Cl- current (GABA-ICl; to 75%), whereas the agent had little effect on the excitatory glutamate response. The reduction in the Gly-ICl was dose-dependent, with a dissociation constant (KD) of 3 mM and a Hill coefficient of 0.96. A non-competitive inhibition was suggested by a double reciprocal plot of the effects of lidocaine on the concentration-response curve of the Gly-ICl. Benzocaine, a neutral local anesthetic at physiological pH, decreased the Gly-ICl more potently than lidocaine, while QX314, a permanently charged quaternary derivative of lidocaine, produced a much smaller inhibition, thereby indicating that the neutral form of local anesthetics is more effective in reducing the Gly-ICl. The depression of the Gly-ICl and GABA-ICl in central neurons may contribute to local anesthetic-induced convulsions.

Action of enflurane on cholinergic transmission in identified Aplysia neurones.

1 Effects of enflurane on the cholinergic transmission in Aplysia neurones were studied by current and voltage clamp methods. Acetylcholine (ACh) evoked three types of postsynaptic responses on different identified neurones: (1) a depolarizing response due to an increase in Na and K conductances (D‐response), (2) a fast hyperpolarizing response due to an increase in Cl conductance (Cl‐response), and (3) a slow hyperpolarizing response due to an increase in K conductance (K‐response). 2 Enflurane altered neither the action potential nor the membrane resistance of the neurones but depressed the three ACh‐induced responses, non‐competitively, in a dose‐dependent manner. The K‐response was less suppressed than the other two. 3 Blockade of the closed state of ion channel was suggested by a reduction in the first ACh response evoked 1 min after administration of enflurane. 4 The anaesthetic facilitated the decay of the neurally evoked e.p.s.c. and i.p.s.c. in suggesting a reduction in the mean open time of the postsynaptic ion channel. 5 It is concluded that enflurane depresses excitatory and inhibitory cholinergic transmission by reducing the postsynaptic currents.

Reduction by thiopental of the slow-channel-mediated action potential of canine papillary muscle

SummaryThe negative inotropic effect of thiopental was examined on the canine papillary muscle. The slow-channel-mediated action potential was induced by 5mM of caffeine in high-K (27 mM) Tyrode solution. The drug reduced the slow action potential and the twitch tension, suggesting a reduction in the slow inward current.

Effects of Ach on slow inward current and tension components of the bullfrog atrium

Effects of acetylcholine (ACh) on the bullfrog atrial muscle were studied by the double gap method under voltage clamped and unclamped conditions. Slight decrease in amplitude and great reduction in duration of the action potential were produced by ACh (10 −6 g/ml) without altering the resting membrane potential. Twitch tensions markedly decreased while contracture tensions due to Na-free or K-excess (100 mM) Ringer solutions were augmented. In voltage clamp experiments, ACh markedly reduced the slow inward current and the I Ca -dependent component of contraction for short depolarizing pulses (less than 100 ms) without affecting time-to-peak tension. For longer pulses, The phasic component was depressed while the tonic component was somewhat enhanced. Steady state activation and inactivation variables of the slow inward current ( d ∞ and f ∞) were not affected by ACh whereas the limiting conductance of Ca (¯g Ca ) was markedly reduced. The time constant of the activation (τd), and that of restoration of calcium conductance were not influenced. It is concluded that ACh exerts its negative inotropic effect on the myocardium not only by shortening of action potential due to increase in potassium conductance but also by decrease in calcium inward current due to reduction in ¯g Ca .

Modulation of miniature inhibitory postsynaptic currents by isoflurane in rat dissociated neurons with glycinergic synaptic boutons.

The effects of a volatile anesthetic, isoflurane, on glycinergic miniature inhibitory postsynaptic currents (IPSCs) were investigated in mechanically dissociated rat trigeminal nucleus neurons with intact glycinergic interneuronal presynaptic nerve terminals. The nystatin-perforated patch recording configuration was used to record the miniature IPSCs under voltage-clamp conditions. Isoflurane shifted in a parallel fashion the glycine (Gly) concentration-response curve of enzymatically dissociated neurons to the left without changing the maximum response. Isoflurane reversibly increased the frequency of the miniature IPSCs and prolonged the decay time constant without affecting the mean amplitude. The increase in the frequency of miniature IPSCs in the presence of isoflurane was also observed in Ca(2+)-free external solution. Thapsigargin prohibited the facilitatory effect of isoflurane on the miniature IPSC frequency. It is concluded that isoflurane increases the Ca(2+) concentration in the glycinergic presynaptic nerve terminal by enhancing the release and/or suppressing the uptake of Ca(2+) into stores.

Effects of methyl p-hydroxybenzoate (methyl paraben) on Ca2+ concentration and histamine release in rat peritoneal mast cells

Mechanisms of methyl p‐hydroxybenzoate (methyl paraben) action in allergic reactions were investigated by measuring the intracellular Ca2+ concentration ([Ca2+]i) and histamine release in rat peritoneal mast cells (RPMCs). In the presence or absence of extracellular Ca2+, methyl paraben (0.1–10 mM) increased [Ca2+]i, in a concentration‐dependent manner. Under both the conditions, methyl paraben alone did not evoke histamine release. In RPMCs pretreated with a protein kinase C (PKC) activator (phorbol 12‐myristate 13‐acetate (PMA) 3 and 10 nM), methyl paraben (0.3–3 mM) induced histamine release. However, a high concentration (10 mM) of the agent did not increase the histamine release. U73122 (0.1 and 0.5 μM), an inhibitor of phospholipase C (PLC), significantly inhibited the methyl paraben‐induced histamine release in PMA‐pretreated RPMCs. U73343 (0.5 μM), an inactive analogue of U73122, did not inhibit the histamine release caused by methyl paraben. In Ca2+‐free solution, PLC inhibitors (U73122 0.1 and 0.5 μM, D609 1–10 μM) inhibited the methyl paraben‐induced increase in [Ca2+]i, whereas U73343 (0.5 μM) did not. Xestospongin C (2–20 μM) and 2 aminoethoxydiphenyl borate (30 and 100 μM), blockers of the inositol 1,4,5‐trisphosphate (IP3) receptor, inhibited the methyl paraben‐induced increase in [Ca2+]i in Ca2+‐free solution. In conclusion, methyl paraben causes an increase in [Ca2+]i, which may be due to release of Ca2+ from storage sites by IP3 via activation of PLC in RPMCs. In addition, methyl paraben possibly has some inhibitory effects on histamine release via unknown mechanisms.