Takashi Akasu

Cyclic AMP regulates an inward rectifying sodium-potassium current in dissociated bull-frog sympathetic neurones.

1. Bull‐frog sympathetic neurones in primary culture were voltage clamped in the whole‐cell configuration. The pipette solution contained ATP (5 mM). 2. A hyperpolarization‐activated sodium‐potassium current (H‐current: IH) was separated from other membrane currents in a nominally calcium‐free solution containing cobalt (2 mM), magnesium (4 mM), barium (2 mM), tetraethylammonium (20 mM), tetrodotoxin (3 microM), apamin (30 nM) and 4‐aminopyridine (1 mM). IH was selectively blocked by caesium (10‐300 microM). 3. The steady‐state activation of IH occurred between ‐60 and ‐130 mV. The H‐conductance was 4.1‐6.6 nS at the half‐activation voltage of ‐90 mV. With the concentrations of potassium and sodium ions in the superfusate at 20 and 70 mM, respectively, the reversal potential of IH was about ‐20 mV. IH was activated with a time constant of 2.8 s at ‐90 mV and 22 degrees C. The Q10 between 16 and 26 degrees C was 4.3. 4. A non‐hydrolysable ATP analogue in the pipette solution did not support IH activation. Intracellular ‘loading’ of GTP‐gamma‐S (30‐500 microM) led to a progressive activation of IH. 5. Forskolin (10 microM) increased the maximum conductance of IH by 70%. This was associated with a depolarizing shift in the half‐activation voltage (5‐10 mV) and in the voltage dependence of the activation/deactivation time constant of IH. 6. Essentially the same results as with forskolin were obtained by intracellular ‘loading’ with cyclic AMP (3‐10 microM) or bath application of 8‐bromo cyclic AMP (0.1‐1 mM), dibutyryl cyclic AMP (1 mM) and 3‐isobutyl‐1‐methylxanthine (0.1‐1 mM). 7. The protein kinase inhibitor H‐8 (1‐10 microM) decreased the peak amplitude of IH. Phorbol 12‐myristate 13‐acetate (10 microM), a protein kinase C activator, was without effect. 8. It is concluded that a voltage‐dependent cation current can be regulated by the basal activity of adenylate cyclase, presumably through protein kinase A, in vertebrate sympathetic neurones.

ATP regulates muscarine-sensitive potassium current in dissociated bull-frog primary afferent neurones.

1. Bull‐frog dorsal root ganglion cells in primary culture were voltage clamped in the whole‐cell configuration. The pipette solution contained ATP (5 mM). 2. Step depolarizations (5‐70 mV, 0.1‐1 s) from a holding potential close to the resting potential (range, ‐64 to ‐79 mV) evoked a non‐inactivating potassium current with properties indistinguishable from those which have been reported for the M‐current of bull‐frog sympathetic neurones. 3. An unhydrolysable ATP analogue APP(NH)P (5 mM), substitute with ATP in the pipette solution, did not support the M‐current activation. 4. Bath application of ATP (30 nM‐30 microM) reduced the amplitude of the M‐current in a concentration‐dependent manner, congruent to 50% inhibition of the current occurring with 1 microM‐ATP. The main effect of ATP was to reduce the maximum M‐conductance without changing the activation and deactivation kinetics of the M‐current. 5. Essentially the same results were obtained with ADP (0.1‐30 microM) and alpha, beta‐methylene‐ATP (10‐30 microM). AMP (10‐100 microM) and adenosine (10‐30 microM) were without effect on the M‐current. 6. The ATP‐induced inhibition of the M‐current was irreversible when an unhydrolysable GTP analogue GTP‐gamma‐S (10‐30 microM) was present in the pipette solution. ATP (3 microM) reduced the amplitude of the M‐current only by about 10% when GDP‐beta‐S (100 microM) was present in the pipette solution. Pre‐treatment of the cells with pertussis toxin (IAP; 500 ng ml‐1) for 24 h at 24 degrees C did not prevent the ATP‐induced M‐current inhibition. 7. Phorbol 12‐myristate 13‐acetate (PMA; 1‐3 microM) reduced the amplitude of the M‐current to about 50%. A reduction in the M‐current amplitude by PMA (3 microM) and ATP (10 microM) was attenuated when staurosporine (200 nM) was present in the pipette solution. Forskolin (10 microM) was without effect on the M‐current. 8. It is concluded that ATP acting at P2 receptors, associated with an IAP‐insensitive GTP‐binding protein, inhibits the M‐current in amphibian primary afferent neurones.

Inward rectifier and low-threshold calcium currents contribute to the spontaneous firing mechanism in neurons of the rat suprachiasmatic nucleus

Intracellular and voltage-clamp studies were carried out to clarify the mechanism for spontaneous firing activity in neurons of the suprachiasmatic nucleus (SCN) of rat hypothalamic brain slices in vitro. SCN neurons displayed spontaneously firing action potentials that were preceded by a depolarizing pre-potential and followed by a short spike after-hyperpolarization (AHP). Injection of inward current with a duration longer than 50 ms resulted in a depolarizing voltage “sag” on hyperpolarizing electrotonic potentials. The inward rectification was depressed by bath application of caesium (1 mM) but not by barium (500 μM). SCN neurons also showed a rebound depolarization associated with spike discharge (anodal break) in response to relaxation of hyper polarizing current injection. The rebound depolarization was reduced by nominally zero calcium. Cadmium (500 μM), cobalt (1 mM) or caesium (1 mM) but not nicardipine also depressed the rebound depolarization. Under voltage-clamp conditions, hyperpolarizing steps to membrane potentials negative to approximately −60 mV caused an inward rectifier current, probably H current (IH), which showed no inactivation with time. Bath application of caesium (1–2 mM) suppressed IH. Caesium (2 mM) depressed the slope of the depolarizing spike pre-potential, resulting in a prolongation of the interspike interval of tonic firing neurons. We conclude that both the inward rectifier current, IH, and the low-threshold calcium current contribute to the spike prepotential of spontaneous action potentials in firing neurons of the rat SCN.

Noradrenaline hyperpolarization and depolarization in cat vesical parasympathetic neurones.

Responses to noradrenaline (NA) applied by superfusion, ionophoresis or pressure pulse were analysed using conventional intracellular recording and voltage‐clamp methods in cat vesical parasympathetic ganglia. NA (1 microM) hyperpolarized 60% of the neurones, depolarized 25%, and produced a biphasic potential, which comprised a membrane hyperpolarization followed by a membrane depolarization, in 10%. About 5% of the neurones did not respond to NA. The NA hyperpolarization was blocked by yohimbine (1 microM), an alpha 2‐adrenoceptor antagonist, whereas the NA depolarization was blocked by prazosin (0.1‐1 microM), an alpha 1‐adrenoceptor antagonist. These data indicated that the NA hyperpolarization was mediated through alpha 2‐adrenoceptors and the NA depolarization through alpha 1‐adrenoceptors. The NA hyperpolarization was accompanied by an increase in conductance, while the NA depolarization was associated with a decrease in conductance measured under manual‐clamp conditions. Similar conductance changes were observed under voltage clamp. NA hyperpolarizations became smaller as the membrane was hyperpolarized and reversed polarity beyond ‐100 mV. NA depolarizations also became smaller at hyperpolarized membrane potentials and reversed polarity around ‐90 mV. The NA responses were enhanced in low‐K media and depressed in high‐K Krebs solution. The NA hyperpolarization was blocked by the Ca antagonists, Cd, Mn and Co. Intracellular injection of EGTA caused a slowly developing, progressive block of the NA hyperpolarization. The NA depolarization was not affected by low Ca concentrations, Ca antagonists or intracellular injection of EGTA. In some neurones the NA depolarization was unmasked in solutions containing Ca antagonists and after intracellular EGTA injection. The NA hyperpolarization was depressed by intracellular injection and extracellular superfusion of Cs but not by TEA. Ba (10‐100 microM) depressed the NA hyperpolarization by 30%. The NA depolarization persisted in the presence of muscarine (10 microM) and was not blocked by Cs or TEA but was depressed 70% by Ba (10 microM). These data are consistent with the hypotheses that alpha 2‐adrenoceptor activation produces a membrane hyperpolarization that is mediated through a Ca‐dependent K conductance, and that alpha 1‐adrenoceptor activation produces a membrane depolarization through closure of a voltage‐insensitive K channel.

Myosin light chain kinase occurs in bullfrog sympathetic neurons and may modulate voltage-dependent potassium currents

A polyclonal antibody against myosin light chain kinase (MLCK) of chicken gizzard recognized a 130 kd peptide of bullfrog sympathetic ganglia as MLCK. MLCK immunoreactivity was confined to the neuronal cell body. A synthetic peptide corresponding to an inhibitory domain of MLCK (Ala783-Gly804) was applied intracellularly to isolated sympathetic neurons during whole-cell recordings of ionic currents. The peptide inhibitor reversibly decreased M-type potassium current (IM) while not affecting A-type of delayed rectifier-type potassium currents. Intracellular application of an active fragment of MLCK enhanced IM, whereas application of an inactive MLCK fragment did not. The results suggest that IM can be modulated by MLCK-catalyzed phosphorylation.

SUBSTANCE P SUPPRESSES GABAA RECEPTOR FUNCTION VIA PROTEIN KINASE C IN PRIMARY SENSORY NEURONES OF BULLFROGS

1. The effects of substance P (SP) and related tachykinins on the function of gamma‐aminobutyric acid‐A (GABAA) receptors were examined in acutely dissociated neurones of bullfrog dorsal root ganglia (DRG) by using whole‐cell voltage‐clamp techniques. 2. Application of SP (10 nM to 1 microM) depressed inward currents produced by GABAA receptor activation (IGABA). Neurokinin A (NKA) and neurokinin B (NKB) also depressed IGABA; the rank order of agonist potency was SP > NKA > NKB. Spantide ([D‐Arg1, D‐Trp7,9,Leu11]SP) and L‐703,606, NK1 receptor antagonists, blocked the SP‐induced depression of IGABA. 3. SP irreversibly depressed IGABA, when neurones were intracellularly dialysed with GTP gamma S. Intracellular application of GDP beta S prevented the SP‐induced depression of IGABA. Pertussis toxin (PTX) did not block the inhibitory effect of SP on IGABA. 4. The depression of IGABA produced by SP was inhibited by H‐7 and PKC(19‐36), protein kinase C (PKC) inhibitors, but not by H‐9 and HA‐1004, protein kinase A inhibitors. IGABA was suppressed by application of sn‐1,2‐dioctanoyl glycerol (DOG), a PKC activator. 5. It is concluded that activation of neurokinin‐1 (NK1) receptors downregulates the function of the GABAA receptor of primary sensory neurones through a PTX‐insensitive G‐protein. PKC may be involved in the transduction pathway of the tachykinin‐induced inhibition of the GABAA receptor.

Slowly inactivating potassium current in cultured bull‐frog primary afferent and sympathetic neurones.

1. Cultured bull‐frog dorsal root ganglion cells were voltage clamped in the whole‐cell configuration. The cells were superfused with a nominally calcium‐free Ringer solution containing tetrodotoxin (3 microM), magnesium (10 mM), cobalt (1 mM), barium (2 mM), 4‐aminopyridine (3 mM) and caesium (2 mM). 2. Step depolarizations (10‐40 mV, 100‐300 ms) from a holding potential close to the rest (typically ‐70 mV) evoked an outward current (IK) followed by an outward tail current. The peak amplitude of the current was reduced to less than 10% by tetraethylammonium (30 mM). 3. IK developed to its peak in 200 ms at ‐30 mV. Tail currents reversed at potentials that changed according to the logarithm of the extracellular potassium concentrations. 4. Tail currents declined to the baseline according to an exponential function of time (tau congruent to 40 ms at ‐60 mV) and its reciprocal time constant increased e‐fold with a 13 mV hyperpolarization. 5. The current inactivated during sustained (1‐20 s) depolarizing pulses according to a single exponential function (tau congruent to 3 s). 6. The peak amplitude of IK at ‐30 mV was progressively increased as the holding potential was made more negative than ‐70 mV reaching the maximum with step depolarizations from ‐120 mV. Reversed phenomenon was observed as the holding potential was made less negative than ‐70 mV. 7. The removal of the steady‐state inactivation occurred along with a single exponential function and the time constant was decreased from 70 ms at ‐70 mV to 10 ms at ‐120 mV. 8. It is suggested that a slowly inactivating potassium current which we called IK in amphibian sensory neurones could be a class of a ‘delayed’ rectifier potassium current. A potassium current with properties indistinguishable from those which have been described for the sensory IK also occurred in cultured bull‐frog sympathetic neurones. 9. Forskolin (1‐30 microM) and 1,9‐dideoxy forskolin (10 microM) reduced the amplitude of IK by up to 85% but these actions were not mimicked by any of 8‐bromo‐cyclic AMP (1 mM), dibutyryl cyclic AMP (1 mM) and 3‐isobutyl‐1‐methylxanthine (1 mM). A hydrophilic forskolin analogue, 7‐O‐hemisuccinyl‐7‐deacetyl forskolin (10 microM), was about one‐tenth as potent as forskolin (10 microM).

ENDOTHELIN MODULATES CALCIUM CHANNEL CURRENT IN NEURONES OF RABBIT PELVIC PARASYMPATHETIC GANGLIA

1 The effects of endothelin were studied, in vitro, on neurones contained in the rabbit vesical pelvic ganglion by use of intracellular and single‐electrode voltage clamp techniques under conditions where sodium and potassium channels were blocked. 2 In the current‐clamp experiments, endothelin (1 μm) caused a depolarization followed by a hyperpolarization of the membrane potential. In the voltage‐clamp experiments, endothelin (0.01–1 μm) caused an inward current followed by an outward current in a concentration‐dependent manner. 3 Membrane conductance was increased during the endothelin‐induced depolarization and inward current. Membrane conductance was decreased during the endothelin‐induced hyperpolarization and outward current. 4 The endothelin‐induced inward and outward currents were not altered by lowering external sodium concentration or raising external potassium concentration. 5 The endothelin‐induced inward current was depressed (mean 72%) in a Krebs solution containing nominally zero calcium and high magnesium. These results suggest that a predominent component of the endothelin‐induced inward current is mediated by calcium ions. 6 The calcium‐insensitive component of the inward current was abolished by a chloride channel blocker, 4‐acetamide‐4′‐isothiocyanostilbene‐2,2′‐disulphonic acid. The mean reversal potential for the calcium‐insensitive component of the inward current was −18 mV. This value is near the equilibrium potential for chloride. Thus, it is presumed that the calcium‐insensitive component of the inward current is carried by chloride ions. 7 Endothelin caused an initial depression followed by a long lasting facilitation of both rapidly and slowly decaying components of high‐threshold calcium channel currents (N‐ and L‐type). 8 In summary, the data show that for neurones in the vesical pelvic ganglia, endothelin causes membrane depolarization and activates an inward current. The ionic mechanisms involve receptor‐operated calcium and chloride currents. Also, endothelin causes an initial depression followed by a long‐lasting facilitation of the voltage‐dependent calcium current.

Volatile anaesthetics inhibit a cyclic AMP-dependent sodium-potassium current in cultured sensory neurones of bullfrog

1 Cultured dorsal root ganglion cells of the bullfrog were voltage‐clamped in the whole‐cell configuration. 2 An adenosine 3′:5′‐cyclic monophosphate (cyclic AMP)‐dependent cationic inward rectifier (IH) was inhibited by bath application of enflurane (0.2‐0.8 mm) and halothane (0.2‐0.5 mm), which thereby induced an outward current at the resting potential, and a membrane hyperpolarization in undamped cells. 3 The main effect of enflurane (0.5 mm) was to displace the steady‐state IH activation curve to a hyperpolarizing direction by about 10 mV, as well as to reduce the maximum H‐conductance to about 20%. 4 Forskolin (1–10 μm), which enhances IH by producing a depolarizing shift in the IH activation curve and increasing the maximum H‐conductance, recruited IH even when the current had already been eliminated by enflurane (1 mm).

Adenosine inhibits the synaptic potentials in rat septal nucleus neurons mediated through pre- and postsynaptic A1-adenosine receptors

Intracellular and voltage-clamp recordings were made from neurons in rat brain slices containing dorsolateral septal nucleus (DLSN), in vitro. Bath application of adenosine (100 microM) produced a hyperpolarization (2-15 mV) in 46% of DLSN neurons (AH-neurons); in the remaining 54% neurons (non-AH-neurons), no hyperpolarization to adenosine was observed. Adenosine (1-300 microM) depressed not only the excitatory postsynaptic potential (EPSP) but also the inhibitory postsynaptic potential (IPSP) and the late hyperpolarizing potential (LHP) evoked by stimulation of the hippocampal CA3 area or the fimbria/fornix pathway in both AH- and non-AH-neurons. In non-AH-neurons, adenosine did not block current responses resulting from glutamate, muscimol or baclofen applied directly to DLSN neurons. In AH-neurons, adenosine partially depressed the baclofen-induced outward current. Adenosine did not block the directly-evoked IPSP (monosynaptic IPSP) as well as the glutamate-induced (hyperpolarizing) postsynaptic potential (PSP) that is mediated by GABA released from interneurons. These results suggest that adenosine does not directly inhibit the release of GABA. The effects of adenosine was mimicked by selective A1-receptor agonists and was blocked by selective A1-receptor antagonists. Pertussis toxin (PTX) blocked the hyperpolarization induced by adenosine or baclofen applied exogenously. Adenosine consistently produced presynaptic inhibition of the EPSP even in DLSN neurons treated with PTX. We conclude that adenosine inhibits neurotransmission between the hippocampus and septum through activation of pre- and postsynaptic A1-receptors which couple with G-proteins of different PTX-sensitivity or with distinct transduction processes at pre- vs. postsynaptic sites.