Nobumasa Hara

Extracellular tumor necrosis factor induces a decreased K+ conductance in an identified neuron of Aplysia kurodai

Recombinant human tumor necrosis factor (rhTNF) was pressure-applied onto the the soma of identified neuron R12 in the Aplysia abdominal ganglion. rhTNF induced a slow inward current (ITNF, 80-100 s in duration, 5-10 nA in amplitude) associated with a conductance decrease. ITNF begins 1-2 s after applying rhTNF and peaks in 5-6 s. ITNF was decreased by hyperpolarization and had a reversal potential of approximately -87 mV (close to the K+ equilibrium potential). Ion substitution and pharmacological experiments suggest that ITNF is due to a decreased K+ conductance and that TNF, a product of macrophages, may form an important link in communications between nervous and immune systems.

Ionic mechanism of the outward current induced by extracellular ejection of interleukin-1 onto identified neurons ofAplysia

The ionic mechanism of the effect of extracellularly ejected recombinant human interleukin-1-beta (rhIL-1) on the membrane of identified neurons R9 and R10 of Aplysia was investigated with voltage-clamp, micropressure-ejection, and ion substitution techniques. Micropressure-ejected rhIL-1 caused a marked hyperpolarization in the unclamped neuron. Clamping the same neuron at its resting potential level (-60 mV) and reejecting rhIL-1 with the same dose produced a slow outward current (I0(IL-1), 20-30 s in duration, 3-5 nA in amplitude) associated with a decrease in input membrane conductance. I0(IL-1) was decreased by depolarization and increased by hyperpolarization. The extrapolated reversal potential of I0(IL-1) was approximately +15 mV. I0(IL-1) was sensitive to changes in the external Na+ concentration but not to changes in K+, Ca2+ and Cl- concentrations, and was resistant to tetraethylammonium (5 mM) and 4-aminopyridine (5 mM). Neither perfusion of the neuron with 50 microM tetrodotoxin nor perfusion with 10 mM Co2+ seawater caused any changes in I0(IL-1). I0(IL-1) was partially reduced by 50 microM ouabain. These results suggest that extracellular IL-1 can induce a slow outward current associated with a decrease in Na+ conductance and the immunomodulator IL-1 can act directly on the nervous system as well as on the immune system.

Nitric oxide induces an increased Na+ conductance in identified neurons of Aplysia

The ionic mechanism of the effects of micropressure ejections of hydroxylamine (HOA) and sodium nitroprusside (SNP), nitric oxide (NO) generators, on the membrane of identified neurons (R9-R12) of Aplysia kurodai was investigated with conventional voltage-clamp, micropressure ejection, and ion-substitution techniques. Micropressure ejection of HOA and SNP onto the neurons caused a marked depolarization in the unclamped neurons. Clamping the same neurons at their resting potential level (-60 mV) and reejecting HOA and SNP with the same dose produced a slow inward current (Ii(HOA) and Ii(SNP), 3-7 nA in amplitude, 15-60 s in duration) associated with an increase in input membrane conductance. Bath-applied hemoglobin (50 microM), a nitric oxide scavenger, almost completely blocked Ii(HOA) and Ii(SNP), and 3-isobutyl-1-methylxanthine (IBMX, 50 microM) prolonged and enhanced both Ii(HOA) and Ii(SNP). An intracellular injection of cyclic guanosine 3,5-monophosphate (cGMP) into the same neurons produced a slow inward current (Ii(cGMP)) which resembled the responses to HOA and SNP, and this current was enhanced in IBMX. Bath-applied methylene blue (10 microM), an inhibitor of guanylate cyclase, significantly reduced Ii(HOA) and Ii(SNP). The inward currents induced by HOA, SNP and cGMP were sensitive to changes in the external Na+ concentration. These results suggest that extracellular NO can induce a slow inward current associated with an increase in Na+ conductance, mediated by an increase in intracellular cGMP.

Activation of single Ca2+-dependent K+ channel by external ATP in mouse macrophages

Single Ca2+‐dependent K+ currents responding to external ATP were recorded from cell‐attached patches on mouse peritoneal macrophages. Extracellularly applied ATP activated an inward single‐channel current with a conductance of 25 pS and a reversal potential of −79 mV (pipette potential, V p) when the pipette contained a 145 mM KC1 solution. The reversal potential was shifted 56 mV positive by a 10‐fold reduction in external (pipette) K+ concentration. The effect of ATP was abolished by either removal of external Ca2+ or treatment with an intracellular Ca2+ chelator, the acetoxymethyl ester of 1,2‐bis (2‐aminophenoxy)ethane‐N,N,N,N‐tetraacetic acid (BAPTA‐AM). This channel has a mean open time of 9.1 ms and open probability was not strongly dependent on V p in the range tested (+ 120 to −30 mV).

The activation of Ca2+-dependent K+ conductance by adrenaline in mouse peritoneal macrophages

Responses to adrenaline in mouse peritoneal macrophages were investigated with perforated and cell-attached patch-clamp recording, and with a combination of the perforated-patch recording and fura-2 fluorescence measurements. Extracellularly applied adrenaline induced a transient outward current (4–10s in duration, 100–500 pA in amplitude) at −40 mV associated with a marked increase in conductance. The adrenaline-induced current [Io (Adr)] reversed polarity near −80 mV. The reversal potential depended distinctly on the external K+ concentration but not on external Cl− concentration. Removal of external Ca2+ did not affect Io(Adr) within 2–4 min but subsequent responses to adrenaline were progressively depressed. In contrast, treatment with an intracellular Ca2+ chelator, the acetoxymethyl ester of 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid completely abolished Io(Adr). Furthermore, Io(Adr) was blocked by bath-applied quinidine and charybdotoxin, but not by tetraethylammonium or apamin. Extracellular application of an α1-adrenoceptor agonist phenylephrine and of noradrenaline mimicked Io(Adr). On the other hand, Io(Adr) was antagonized by a non-selective α-adrenoceptor antagonist phentolamine (0.2 μM) and an α1-adrenoceptor antagonist prazosin (0.2 μM), but was not affected by an α2-adrenoceptor antagonist yohimbine (1 μM) or a β-adrenoceptor antagonist propranolol (1 μM). Cell-attached single-channel recordings with the pipette solution containing 145 mM KCl revealed the activation of single-channel currents with a conductance of 40 pS during application of adrenaline outside the patch. Parallel measurements of membrane current and fura-2 fluorescence in the same cell demonstrated a correlation between the rise in [Ca2+]i and an increase in K+ conductance. Therefore, it is concluded that adrenaline activates a Ca2+-dependent K+ conductance by release of Ca2+ from internal stores through an activation of an α1-adrenoceptor.

Tumor necrosis factor reduces the ACh-induced outward current in identified Aplysia neurons

Effects of extracellularly applied recombinant human tumor necrosis factor (rhTNF) on the acetylcholine (ACh)-induced K+ current recorded from identified neurons (R9 and R10) of Aplysia kurodai were investigated with conventional voltage-clamp and pressure ejection techniques. Bath-applied rhTNF (200-500 U/ml) reduced the ACh-induced current in the neurons without affecting the holding current and resting membrane conductance. The suppressing effect of rhTNF on the current was completely reversible. Inhibition by rhTNF was non-competitive. Heat-inactivated rhTNF was without effect. Our results suggest that the immunomodulator TNF can act on the ACh receptor in the nervous system.

Activation of K+ current in macrophages by platelet activating factor.

Puff application of platelet activating factor (10(-8) M) onto peritoneal macrophages from thioglycollate-stimulated mice induced an outward current at a holding potential of -63 mV. The current was suppressed by an antagonist Y-24180 but not by CV-3988. Charybdotoxin (10(-6) M) suppressed the current. Reversal potentials were dependent on external K+ concentrations. The current was not suppressed in Ca(2+)-free EGTA-containing solution but was completely abolished in BAPTA-AM containing solution. These results suggest that platelet activating factor activates a Ca(2+)-dependent K+ channel.

Reduction of the acetylcholine-induced K+ current in identifiedAplysia neurons by human interleukin-1 and interleukin-2

Summary1.Effects of bath-applied recombinant human interleukin-1 (rhIL-1) and interleukin-2 (rhIL-2) on the acetylcholine (ACh)-induced K+ current recorded from identified neurons (R9 and R10) ofAplysia kurodai were investigated with voltage-clamp and pressure ejection techniques.2.Bath-applied rhIL-1 and rhIL-2 (10–40 U/ml) reduced the ACh-induced current in the neurons without affecting the resting membrane conductance and holding current.3.The suppressing effects of these cytokines on the current were completely reversible.4.Heat-inactivated rhIL-1 and rhIL-2 were without effect.5.These results suggest that the immunomodulators, IL-1 and IL-2, can modulate the ACh-induced response in the nervous system.

Induction of two K+ currents by complement component C5a in mouse macrophages

Puff application of complement component C5a (5 x 10(-8) M) onto peritoneal macrophages from thioglycollate-stimulated mice induced two kinds of outward current at a holding potential of -68 mV, a slowly-rising sustained outward current and a spike-like transient outward current. Quinidine (2 x 10(-4) M) and tetraethylammonium (10(-2) M) partially suppressed both types of outward current. Charybdotoxin (2 x 10(-6) M) markedly suppressed the spike-like outward current. Reversal potentials in bath solutions of different external K+ concentrations were dependent only on K+ concentrations. The transient current was not suppressed in Ca(2+)-free EGTA-containing solution, but was completely abolished in BAPTA-containing solution. One kind of single channel responding to C5a, which has a single-channel conductance of 29 pS, was recorded from cell-attached patches. These results suggest that C5a activates a Ca(2+)-dependent and another type of K+ current.

The neuropeptide, neuromedin C, activates a potassium current in mouse macrophages

Neuromedin C (NmC) induced an outward current (I o(NmC)) in macrophanges. Reversal potentials were dependent on external K+ concentrations ([K+]o) and independent of [Cl−]o. Tetraethylammonium (TEA) and quinidine effectively suppressed I o(NmC). Charybdotoxin (ChTX) and apamin had little effect. I o(NmC) was abolished in Ca2+‐free EGTA‐containing solution. These results suggest that NmC activates a Ca2+‐dependent K+ current (I K.Ca) and can modulate activities in macrophages.