Masashi Sawada

Analysis of hyperpolarizations induced by glutamate and acetylcholine on Onchidium neurones

1. Four giant neurones, designated G‐H cells, in the right pleural ganglion of the marine pulmonate mollusc, Onchidium verruculatum, showed characteristic membrane hyperpolarization during applications of either acetylcholine (ACh) or L‐glutamate. In the presence of ACh the membrane was hyperpolarized only transiently, while in the presence of glutamate the response was maintained. Significant increases in membrane conductance accompanied the changes in membrane potential.

Enhancement of phagocytosis by dynorphin A in mouse peritoneal macrophages

The effects of the opioid peptide dynorphin A (DynA) on phagocytosis in peritoneal macrophages was examined by flow cytometry (FCM). DynA enhanced phagocytosis in a dose-dependent manner. Leucine-enkephalin (Leu-Enk), methionine-enkephalin (Met-Enk), beta-neo-endorphin (beta Neo-End), DynA(9-17) and DynA(13-17) had no such activity. Alpha-Neo-endorphin (alpha Neo-End), dynorphin B (DynB), DynA(1-13) and DynA(6-17) enhanced phagocytosis less effectively than DynA. Naloxone did not inhibit the enhancement of phagocytosis induced by DynA. Unstimulated control phagocytosis was partially suppressed in Ca2+-free EGTA-containing solution and even in this solution DynA enhanced phagocytosis. However, the enhancement by DynA was suppressed in EGTA- and BAPTA-AM-containing Ca2+-free solution. The present study showed that enhancement of phagocytosis by DynA was independent of extracellular Ca2+ ([Ca2+]o) and dependent on intracellular Ca2+ ([Ca2+]i). The present results support DynA being one of the mediators from the nervous system that modulates the immune system.

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.

Enhancement of phagocytosis by calcitonin gene-related peptide (CGRP) in cultured mouse peritoneal macrophages

Calcitonin gene-related peptide (CGRP) is widely distributed in sensory neurons and nerve fibers. To clarify the function of CGRP on the immune system, the effect of CGRP on phagocytosis by peritoneal macrophages was examined by means of flow cytofluorometry. CGRP enhanced phagocytosis of latex beads in a dose-dependent manner. Because the phosphodiesterase inhibitor 3-isobutyl, 1-methylxanthine (IBMX) enhanced the CGRP-induced enhancement of phagocytosis, the enhancement might be mediated by cAMP. In the presence of mannan, the phagocytosis was suppressed and the CGRP-induced enhancement was also blocked, suggesting that mannose receptors on macrophages were involved in mediating the phagocytosis of latex beads, and CGRP enhanced the mannose receptor-mediated phagocytosis. The present results indicate that CGRP can modulate the function of macrophages in nerve terminals of sensory neurons during the development and maintenance of inflammation.

Enhancement of phagocytosis in mouse macrophages by pituitary adenylate cyclase activating polypeptide (PACAP) and related peptides

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) and related peptides on phagocytosis of fluorescent latex beads by mouse peritoneal macrophages were examined using flow cytometry (FCM). PACAP38, PACAP27 and vasoactive intestinal peptide (VIP) enhanced phagocytosis in a dose-dependent manner. Relative potencies of related peptides at a concentration of 10(-6) M were PACAP38 > PACAP27 > VIP > secretin > glucagon > (peptide with NH2-terminal histidine and COOH-terminal methionine amide, in short PHM). PACAP(6-38) was as effective as PACAP38. PACAP(6-27) enhanced phagocytosis more effectively than did PACAP27. PACAP(28-38) slightly enhanced phagocytosis. The present result suggest that PACAP38 is one of the mediators that the nervous system uses to modulate the immune system.

Induction of outward current by orexin-B in mouse peritoneal macrophages

To define effects of novel feeding regulating peptides, orexins, in immunocompetent cells, ion channel activity in mouse peritoneal macrophages was analyzed by the perforated patch‐clamp method. Orexin‐B (OX‐B) induced an outward current at smaller holding potentials than K+ equilibrium potentials. Reversal potentials of OX‐B induced current were dependent on external K+ concentrations but not on external Cl− concentration. Orexin‐A is less effective than OX‐B. Quinine blocked the outward current and tetraethylammonium partially suppressed the current. These results suggest that OX‐B can modulate macrophage functions through the activation of Ca2+‐dependent K+ channels.

Enhancement of phagocytosis by corticostatin I (CSI) in cultured mouse peritoneal macrophages.

Corticostatin I (CSI) is one of the corticostatic peptides which inhibit ACTH-stimulated steroidogenesis. To clarify the function of CSI on the immune system, the effect of CSI on phagocytosis by peritoneal macrophages was examined by means of flow cytofluorometry. In the presence of Ca2+ and Mg2+, CSI enhanced phagocytosis of latex beads in a dose-dependent manner. Unstimulated phagocytosis in physiological solution consisted of Ca2+ and Mg(2+)-dependent and -independent phagocytosis. Divalent cations-independent phagocytosis was sensitive to CSI. Present results suggest that the enhancement of phagocytosis by CSI may be one of the mechanisms modulating the immune response regarding infection and inflammation. Present study also showed that one of defensin HNP-1 enhanced phagocytosis.

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.

Suppression of phagocytosis by adrenocorticotropic hormone in murine peritoneal macrophages.

Phagocytosis of latex beads by peritoneal macrophages was examined by means of flow cytometry (FCM). This assay revealed that adrenocorticotropic hormone (ACTH) suppressed phagocytosis in a dose-dependent manner. ACTH (1-24) was more suppressive than ACTH (1-39). Control phagocytosis was partially suppressed in Ca(2+)-free solution. Phagocytosis was suppressed by ACTH in this solution to the same degree as in the normal solution. Suppression by ACTH was reduced in phosphodiesterase inhibitor-containing solution. These results suggest that (1) ACTH suppresses extracellular Ca(2+)-dependent and -independent phagocytosis, (2) the suppression is not mediated by cAMP and (3) the inhibition of macrophage phagocytosis by ACTH is one of the mechanisms that modulate immune responses in stressful situations.