Mitsuyuki Ichinose

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.

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.

Role of protein phosphatases in the modulation of neuronal membrane currents

Although much is known regarding the modulation of ion channels through protein phosphorylation, little is known about their dephosphorylation by protein phosphatases (PrPs). To address this issue we examined the effects of a phosphatase inhibitor, okadaic acid (OA), and protein phosphatases obtained from rabbit skeletal muscle on steady-state membrane current in voltage-clamped sensory neurons of Aplysia. In addition, we examined the ability of OA and PrPs to modulate serotonin (5-HT)- and cAMP-induced inward currents and an opposite outward current induced by the peptide FMRFamide. The results suggest that: (1) some basal level of phosphorylation regulates membrane channels even in the absence of physiological stimulation; (2) the 5-HT-, cAMP-, and FMRFamide-induced responses are highly regulated by PrPs; and (3) the FMRFamide-induced outward current may be due at least in part to the activation of PrPs.

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.

Microcystin-LR, a potent protein phosphatase inhibitor, prolongs the serotonin- and cAMP-induced currents in sensory neurons of Aplysia californica.

Microcystin-LR (MCYST-LR), a hepatotoxin produced by cyanobacteria, inhibited purified protein phosphatases (PrPs) from rabbit skeletal muscle and the enzymes from Aplysia with an IC50 of approximately 10(-10) M. MCYST-LR also prolonged both serotonin- (5-HT) and cyclic adenosine monophosphate-induced inward currents in sensory neurons of Aplysia. These results, which are consistent with inhibition of Aplysia PrPs, indicate that MCYST-LR may be a useful probe to elucidate the function of PrPs in neural tissues.

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.

Nitric oxide inhibits the dopamine-induced K+ current via guanylate cyclase in aplysia neurons

Nitric oxide (NO) is produced by the enzyme nitric oxide synthase (NOS) and has been implicated in inter‐ and intracellular communication in the nervous system. The present study was undertaken to assess the effects of sodium nitroprusside (SNP) and hydroxylamine (HOA), NO donors, on a dopamine (DA)‐induced K+ current in identified Aplysia neurons using voltage‐clamp and pressure ejection techniques. Bath‐applied SNP (10–25 μM) reduced the DA‐induced K+ current without affecting the resting membrane conductance and holding current. The DA‐induced K+ current also was inhibited by the focal application of 200 μM HOA to the neuron somata. The DA‐induced K+ current suppressing effects of SNP and HOA are completely reversible. Pretreatment with 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 1 μM), a specific inhibitor of NO‐stimulated guanylate cyclase, and hemoglobin (50 μM), a nitric oxide scavenger, decreased the SNP‐induced inhibition of the DA‐induced current. In contrast, intracellular injection of 1 mM guanosine 3′,5′‐cyclic monophosphate (cGMP) or bath‐applied 3‐isobutyl‐1‐methylxanthine (IBMX; 50 μM), a nonspecific phosphodiesterase inhibitor, inhibited the DA‐induced current, mimicking the effect of the NO donors. These results demonstrate that SNP and HOA inhibit the DA‐induced K+ current and that the mechanism of NO inhibition of the DA‐induced current involves cGMP‐dependent protein kinase. J. Neurosci. Res. 50:450–456, 1997.

Amyloid β proteins reduce the GABA-induced Cl− current in identified Aplysia neurons

The amyloid beta protein (A beta P) is the major component of the amyloid deposition which characterizes Alzheimers disease. Effects of extracellularly applied A beta P on the gamma-aminobutyric acid (GABA)-induced Cl- current recorded from identified neurons (R9 and R12) of Aplysia kurodai were investigated with conventional voltage-clamp and pressure ejection techniques. Focal application of 100 nM A beta P (1-40) reduced the GABA-induced hyperpolarization at resting membrane potential and the GABA-induced Cl- current in the neurons held at -50 mV. Bath-applied 100 nM A beta P fragments (1-40) and (25-35) but not A beta P (1-16) inhibited the GABA-induced Cl- current as well as muscimol-induced Cl- currents in the neurons without affecting the resting membrane conductance or holding current. These results suggest that A beta P may increase neuronal excitability by inhibiting GABA-induced Cl- current in the neurons of mammalian central nervous system.

ACTIVATION OF OUTWARD CURRENT BY PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE IN MOUSE MICROGLIAL CELLS

In order to investigate the interaction between the nervous and immune systems, we have analyzed the effect of one of the neuropeptides, pituitary adenylate cyclase activating polypeptide (PACAP), on microglia cells by the patch‐clamp method. Puff application of PACAP38 onto mouse microglial cells induced an outward current in a dose‐dependent manner. Reversal potentials of the outward current were dependent on external K+ concentrations ([K+]o) and independent of [Cl−]o. Ion channel blockers of potassium currents, quinine (1 mM), tetraethylammonium (TEA, 20 mM) and 4‐aminopyridine (4‐AP, 5 mM), suppressed the outward current with a potency order of quinine>TEA>4‐AP. PACAP27 also induced outward current less effectively than PACAP38. A fragment of PACAP38 [PACAP(6‐38)], known as an inhibitor for PACAP38, suppressed the outward current. These data suggest that PACAP38 activates a quinine‐sensitive K+ outward current and modulates activities in microglia. They indicate that the immune system in the brain can be modulated by neurotransmitters, the mediators of neurons. J. Neurosci. Res. 51:382–390, 1998. © 1998 Wiley‐Liss, Inc.