Biomedical subjects
Publications and source records attributed to I Tatsuno.
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We have investigated the effects of pituitary adenylate cyclase-activating polypeptide with 38 residues (PACAP38) on glutamate-induced neuronal cell death in rat-cultured cortical neurons. The rat-cultured neurons were obtained from E17 day-old embryos and cultured in a chemically defined medium without serum for 10 days, after which more than 95% of the cells were stained by a specific antibody against MAP-2, a specific marker for neurons. The number of viable neurons was identified by the mitochondrial conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to formazan, which was detected by the associated change in optical density at 570 nm. Glutamate-induced neuronal cell death was suppressed by PACAP38 at concentrations as low as 10(-13) M, and at 10(-11) M maximally suppressed half of the amount of glutamate-induced cell death seen in a control situation (no PACAP38). The dose-response curve was bell-shaped. Dibutyryl cAMP (dbcAMP) also increased the number of neurons that were protected from damage with a bell-shaped dose-response curve suggesting that PACAP exerts its neuroprotective effect through the activation of a cAMP signal transduction system. However, cAMP accumulation in the media of neurons was stimulated by PACAP38 at concentrations as low as 10(-11) M, a much higher concentration than the minimal effective dose of PACAP38 required for protection against glutamate-induced neuronal cell death. Among the three neuropeptides of PACAP38, arginine vasopressin (AVP) and C-type natriuretic peptide (CNP), only PACAP38 exhibited a neurotrophic effect in the glutamate-induced neuronal cell death at the indicated concentrations. These data indicate that PACAP38 is one of the more important neuroprotective factors. The kind of intracellular signal transduction system involved in the neuroprotective effect of PACAP38 still remains to be established.
Many transmembrane receptors are oligomeric proteins. Binding of a ligand may alter the oligomeric state of the receptor, induce structural changes within the oligomer, or both. The bacterial aspartate chemoreceptor Tar forms a homodimer in the presence or absence of ligands. Tar mediates attractant and repellent responses by modulating the activity of the cytoplasmic kinase CheA. In vivo intersubunit suppression was used to show that certain combinations of full-length and truncated mutant Tar proteins complemented each other to restore attractant responses to aspartate. These results suggest that heterodimers with only one intact cytoplasmic domain are functional. The signaling mechanism may require interactions between dimers or conformational changes within a single cytoplasmic domain.
It has been reported that pituitary adenylate cyclase activating polypeptide (PACAP) is a sensory neuropeptide and that it is contained in capsaicin sensitive fibers. PACAP occurs as two variants, PACAP27 and the C-terminally extended PACAP38. We examined the effect of intrathecally administered PACAP27 and PACAP38 on the rat formalin test which is considered to be a model of animal pain induced by inflammation. Fifty microliters of 5% formalin was injected subcutaneously in the plantar surface of the right hind paw. To analyze the effect of PACAP27 and PACAP38, the instances of spontaneous flinching were counted. Administration of either PACAP27 or PACAP38 resulted in a monotonically dose-dependent reduction in the instances of flinching behavior. These results suggested that PACAP may play an important role in inhibiting the transmission of the nociceptive information evoked by inflammation.
We investigated the effects of pituitary adenylate cyclase-activating polypeptide (PACAP38) on cytosolic free calcium ([Ca2+]i) at a single cell level in both type-1 and type-2 cultured rat astrocytes using a calcium-sensitive fluorescent dye, fura-2. Type-1 astrocytes were relatively silent during the 20 min observation of baseline [Ca2+]i and PACAP38 did not alter [Ca2+]i in type-1 astrocytes at concentrations up to 1 microM. In contrast, type-2 astrocytes could be divided into three subtypes (silent type, randomly-firing type and oscillatory type) based on the observation of baseline [Ca2+]i. Of 166 type-2 astrocytes tested, 145 (87.4%) were the silent type (stable basal [Ca2+]i levels) and 13 (7.8%) were the randomly-firing type (random increases in [Ca2+]i). PACAP38 could stimulate [Ca2+]i in subpopulations of all three subtypes. In the silent type-2 astrocytes (4 experiments with 255 cells/experiment), 54.4 +/- 3.6% of the total number responded to PACAP38. The half maximal concentration (ED50) of PACAP38 was 2.89 +/- 1.89 nM. Removing Ca2+ from the superfusion media did not alter the PACAP38-induced increase of [Ca2+]i. Neither 1-30 microM of forskolin nor 1-10 mM of dibutyryl cyclic AMP increased [Ca2+]i in the same type-2 astrocytes which responded to PACAP38. These findings suggest that PACAP increases [Ca2+]i in type-2, not type-1, rat astrocytes by mobilizing Ca2+ from intracellular stores, and that this action is not linked to activation of the cyclic AMP second messenger system.
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Interleukin-6 (IL-6) is a pleiotropic cytokine that is produced by astrocytes and microglia and may act as a trophic factor in the nervous system. These experiments were intended to identify neuroactive agents that regulate IL-6 production in primary cultured rat astrocytes. Addition of either lipopolysaccharide (LPS) or human recombinant interleukin-1 beta (IL-1 beta) to rat astrocytes in culture stimulated IL-6 secretion. However, LPS was significantly more efficacious in eliciting IL-6 production compared to IL-1 beta. Co-addition of the specific IL-1 receptor antagonist (IL-1ra) completely inhibited IL-1 beta-induced IL-6 secretion but did not affect LPS-stimulated IL-6 production during a 6 h incubation period. Two neuroactive peptides, pituitary adenylate cyclase activating polypeptide (PACAP38) and vasoactive intestinal peptide (VIP), stimulated IL-6 production either alone or in combination with IL-1 beta. PACAP38 was significantly more potent in stimulating IL-6 compared to VIP. Results from these experiments indicate that LPS is an effective inducer of IL-6 production in rat astrocytes. This effect of LPS is independent of astrocyte IL-1 production since the IL-1ra was unable to inhibit LPS-stimulated IL-6 secretion. Also, the neuropeptides PACAP38 and VIP are potential secretagogues for IL-6 secretion, and both peptides synergize with IL-1 to stimulate IL-6 secretion in rat astrocytes.
A 42-year-old man with a remittent fever was found to have both para-aortic and hepatic tumors with generalized lymphadenopathy. The pathological findings from biopsy specimens from the para-aortic lymph node and hepatic tumor by laparotomy and from left supraclavicular lymphadenectomy showed undifferentiated carcinoma. However, the location of the primary lesion could not be determined. Chemotherapy temporarily reduced the size of the metastatic lymph nodes and the hepatic tumor and also suppressed the remittent fever. Fifteen months after onset, massive polyclonal hypergammapathy developed together with plasmacytosis (30% plasmacytes) in bone marrow, consisting of normal mature plasmacytes. Among cytokines, including interleukin (IL) 1 beta, IL-3, IL-4, IL-6, and tumor necrosis factor alpha, a high value of IL-6 was detected in the serum. Postmortem pathological examination showed the scirrhous type of gastric carcinoma with specific staining by polyclonal antibody against human IL-6. To our knowledge, this is the first report of a gastric carcinoma producing IL-6 associated with fever, hypergammaglobulinemia, and plasmacytosis in bone marrow.
The developmental changes of PACAP and its receptor in the rat brain were investigated. The levels of PACAP and its receptor in the brain were determined by radioimmunoassay (RIA) and radioreceptor assay, respectively. The [125I]PACAP27 binding to the whole brain membrane preparations from embryonic day (E)18 rats was specific, rapid, and temperature dependent. Scatchard plot analysis revealed only a single class of receptor with Kd for PACAP27 = 1.015 +/- 0.049 nM, Kd for PACAP38 = 0.105 +/- 0.003 nM, and Bmax for both ligands = 8.64 +/- 1.29 pmol/mg protein. The Kd for PACAP38 was significantly lower than that for PACAP27. The binding site for PACAP was detected as early as E14. The Bmax for PACAP gradually increased throughout development to reach a plateau between 1 and 4 months old (MO). The content of PACAP38 in the whole brain was detected by RIA as early as E14. The concentration of PACAP38 in the whole brain, hippocampus, neocortex, and cerebellum gradually increased throughout the pre- and postnatal periods to reach a peak at 1 MO and then tended to decrease between 1 and 4 MO. On the other hand, PACAP38 concentration in the diencephalon rapidly increased between E18 and the birth (P0), and then increased more slowly throughout the postnatal period to reach a peak at 1 MO. In view of a neurotrophic activity of PACAP, these findings may support the notion that PACAP plays a regulatory role in neuronal growth and development.
The serine and aspartate chemosensory receptors (Tsr and Tar) of Escherichia coli have two membrane-spanning regions TM1 and TM2. To investigate their roles in transmembrane signalling, we constructed two chimeric receptors from Tsr and Tar with heterologous combinations of TM1 and TM2: the N-terminus of one receptor, including TM1 and the periplasmic domain, was fused to the C-terminus of the other, beginning with TM2. Both of the chimeric receptor genes rescued the chemotactic defect of a receptorless E. coli strain, indicating that the chimeric receptors are functional. Their apparent affinities for the specific ligands were the same as those of Tsr or Tar. Therefore, as far as transmembrane signalling abilities are concerned, the TM2 regions of Tsr and Tar are interchangeable, suggesting that sequence-specific interaction between TM1 and TM2 may not be required for the signal transmission across the membrane. The cells expressing either of the chimeric receptors, however, showed 'smooth', biased, basal swimming patterns. Moreover, they adapted quickly after stimulation with the repellent glycerol. This rapid adaptation was observed even in the methyltransferase-defective strain. Therefore, exchange of TM2 might impose structural constraints on the chimeric receptors that stabilize conformations which elicit smooth swimming.
Recently, pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalamus and it was shown to stimulate adenylate cyclase in rat pituitary cells, neurons, and astrocytes. PACAP exhibits a 68% amino acid sequence homology with vasoactive intestinal peptide (VIP); however, it is 1000 times more potent than VIP in stimulating adenylate cyclase. In view of the wide distribution of PACAP and its receptor in the central nervous system, PACAP is likely to act as a neurotransmitter or neuromodulator as well. In the present study, we investigated the effects of PACAP38 on cytosolic-free calcium concentrations ([Ca2+]i) and compared these effects with those of VIP in cultured rat hippocampal neurons. Calcium concentrations, at the single cell level, were measured using fura-2, a calcium sensitive fluorescent dye, and fura-2-loaded neurons were continuously superfused at 37 C and viewed under an inverted microscope. Images of these neurons were recorded at 10-sec intervals by a video camera equipped with an Argus-50/CA system which controls the image acquisition and display. [Ca2+]i was quantitated from the intensities of fluorescence of the cells at two excitation wavelengths of 340 and 380 nm. The ratio of the intensities of emitted fluorescence (340/380 nm) was calibrated to determine [Ca2+]i. PACAP38 (0.1 nM) increased [Ca2+]i in some hippocampal neurons. As the concentration of peptide was increased from 0.1 to 10 nM, the accumulated number of hippocampal neurons responding to PACAP38 progressively increased and reached a plateau at 10 nM. Total neurons (33.0 +/- 5.3%, n = 4; 502 neurons) were found to respond to 100 nM PACAP38. The half-maximal concentration (ED50) of PACAP38 was 2.60 +/- 0.77 nM. Typically, 60-90 sec after the addition of PACAP38 (10 nM), [Ca2+]i increased from basal levels of 50-100 to 150-300 nM. VIP also increased [Ca2+]i, but required 1 microM or higher concentration for a considerable number of cells to respond. The number of hippocampal neurons responding to VIP at 1 microM was 28.9 +/- 9.8% (n = 4; 442 cells) which was comparable to the population of neurons responding to 10 nM PACAP38. The ED50 for VIP was 0.68 +/- 0.38 microM which was approximately 260 times higher than the ED50 for PACAP38. Neither 1-10 microM nitrendipine, a L-type voltage-dependent Ca2+ channel blocker, or 1 microM omega-conotoxin GVIA, a N-type voltage-dependent Ca2+ channel blocker, altered the PACAP-induced Ca2+ increment. Removal of Ca2+ from the superfusion media did not influence the PACAP38-induced increase of [Ca2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)
In the present study we performed experiments to reveal the role of 5-lipoxygenase products of arachidonic acid in cell-to-cell interaction between rat peritoneal macrophages and spleen natural killer (NK) cells. Peritoneal macrophages were found to significantly enhance NK cell activity. It was found that the enhancing activity of macrophages was significantly inhibited by the ingestion of highly purified eicosapentaenoic acid-ethyl ester (EPA-E). But direct cytotoxic action of macrophage on target cells (YAC-1) was unchanged by the ingestion of EPA-E. In addition, the depressed enhancing activity of EPA-enriched macrophages (EPA macrophages) was partially, but significantly, restored by the addition of either 10(-10) M leukotriene B4 (LTB4) or 10(-12) M 5-hydroperoxyeicosatetraenoic acid (5-HPETE). The partial restoring effect of 10(-10) M LTB4 was further significantly enhanced by the addition of 5-HPETE. In contrast 5-hydroxyeicosatetraenoic acid (5-HETE) had no modulatory effect on the depressed activity of EPA macrophages. LTB4, 5-HPETE, and 5-HETE had no direct effect on NK cell activity itself. Significantly less amounts of LTB4 and 5-HETE were produced in EPA macrophages as compared with that in control macrophages. The present study indicates that macrophage modulatory action on NK cells can be partly mediated by LTB4 and 5-HPETE, which are produced in macrophages.
The goal of these experiments was to identify and characterize binding sites in the rat hypothalamus for the peptide, pituitary adenylate cyclase activating polypeptide (PACAP). The 27 amino acid form of PACAP (PACAP27) was used as the radiolabeled ligand in these experiments. Binding of [125I]PACAP27 to hypothalamic membrane preparations was rapid, reversible on addition of unlabeled peptide, and at least partially regulated by GTP. Nonhydrolyzable GTP analogs, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), guanosine-5'-(2-thiodiphosphate) (GDP beta S), and guanylylimidophosphate (GppNHp) also displaced [125I]PACAP27 binding to hypothalamic membrane preparations in a dose-dependent manner. The order of potency for the three analogs was GTP gamma S greater than GDP beta S greater than GppNHp. Both forms of the peptide, PACAP27 and PACAP38, were highly potent in displacing bound [125I]PACAP27, whereas VIP or PACAP(1-23) were unable to displace binding at concentrations of up to 500 nM. Scatchard analysis of the PACAP27 and PACAP38 displacement curves revealed that the fit of both curves was consistent with a single class of high-affinity binding sites, although the site exhibited a greater affinity for PACAP38 compared with PACAP27 (PACAP27 Kd = 1452 +/- 59 pM; PACAP38 Kd = 175 +/- 13 pM; Bmax 23.2 +/- 1.1 pmol/mg protein). The possibility of the existence of a class of binding sites with extremely low affinity cannot be discounted. After covalent cross-linking of [125I]PACAP27 with its receptor, the molecular weights of the complexes were estimated by electrophoresis and autoradiography. A major band of 60 Kd was evident when membranes were incubated with VIP or PACAP(1-23). Previous incubation with unlabeled PACAP27 or PACAP38 eliminated visualization of this band. These results suggest that a specific, high-affinity binding site for PACAP27 is present in rat hypothalamus, and that this site shows a greater affinity for PACAP38 compared with PACAP27. The molecular weight of the peptide-receptor complex is 60,000 kDa, and therefore the receptor itself has an apparent molecular weight 57,000.
Interleukin 1 (IL-1) is a multifunctional cytokine produced by numerous cell types, including cells of the central nervous system (CNS). In the CNS, IL-1 produced by glia is thought to support trophic functions after brain injury. However, little is known about whether the IL-1 receptor (IL-1R) is expressed in brain cells and how these receptors might be regulated. Analysis of IL-1R expression in the human glioblastoma cell line U-87 MG indicated the presence of a specific, saturable, and high-affinity (dissociation constant = 104 +/- 14 pM) binding site, which was of moderately high density (1,228 +/- 156 sites/cell). Incubation of U-87 MG cells with cortisol or dexamethasone for as little as 6 h resulted in an upregulation of IL-1R expression, which could be blocked by coincubation with cycloheximide or actinomycin D. IL-1 beta downregulated the expression of its own binding site. Upregulation of the IL-1R by glucocorticoids (GCs) appeared to be coupled to the release of interleukin 6 (IL-6), since IL-1 was significantly more potent in inducing IL-6 release in U-87 cells that were preincubated in the presence of GCs compared with cells incubated in the absence of GCs. These results suggest that IL-1 acts on glial cells via a high-affinity receptor and indicate that GCs may amplify or prolong the actions of IL-1 in the CNS.
Two novel polypeptides known as pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38) and a shorter form of the peptide corresponding to the N-terminal 27 residues (PACAP27) were isolated from ovine hypothalamus. The N-terminal 28 residues of PACAP show 68% homology with vasoactive intestinal peptide (VIP). VIP has been reported to have specific binding sites in lymphocytes and inhibit mitogen-stimulated lymphocyte proliferation through a receptor-mediated stimulation of cAMP-dependent protein kinase. Using concanavalin A-induced proliferation of murine splenocytes as a model system, we now report that both PACAP38 and PACAP 27 can inhibit the proliferation of these cells in the same dose-dependent manner as VIP. The minimal effective concentration of the PACAPs was 10(-10)-10(-9) M. However, neither PACAP affected lipopolysaccharide-induced proliferation of murine splenocytes. The binding of [125I]PACAP27 to these splenocytes was rapid, time dependent, reversible, and proportional to the numbers of murine splenocytes. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites. The dissociation constant (Kd) was 0.86 +/- 0.24 nM and the maximal binding capacity (Bmax) was 1.13 +/- 0.39 fmol/10(6) cells for the high affinity binding site. The low affinity binding site had a Kd of 0.13 +/- 0.03 microM with a Bmax of 73.5 +/- 9.5 fmol/10(6) cells. PACAP38 and VIP displaced the binding of [125I]PACAP27 in the same manner as PACAP27 and Scatchard analyses indicated the presence of two classes of binding sites with Kd and Bmax similar to those for PACAP27. Furthermore, when [125I]VIP was used as a radiolabeled ligand, PACAP27 and PACAP38 displaced the [125I]VIP binding to the same degree as unlabeled VIP. Scatchard analysis indicated that there was no significant difference of the Kd or Bmax between PACAP and VIP. Taken together, these data suggest that PACAPs bind to a site similar or identical to that used by VIP which inhibit the proliferation of murine splenocytes induced by concanavalin A.
Interleukin 6 (IL-6) production was shown to be stimulated by vasoactive intestinal peptide via cAMP dependent signal transduction pathway in the pituitary. We were interested in whether other hypothalamic neuropeptides, which activate adenylate cyclase in the pituitary, also stimulate pituitary IL-6 production. Whereas vasoactive intestinal peptide was effective in stimulating pituitary IL-6 production only at concentrations of 10(-6) M or higher, pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38) and calcitonin gene-related peptide (CGRP) at concentrations from 10(-10) to 10(-9) M significantly stimulated IL-6 production. Similar effective concentrations of each peptide were required for activating adenylate cyclase, as measured by extracellular cAMP accumulation. H89, a specific inhibitor of cAMP dependent protein kinase (protein kinase A), inhibited IL-6 production stimulated by PACAP38, CGRP, and (Bu)2cAMP. However, H89 failed to inhibit the IL-6 production stimulated by lipopolysaccharide, a ligand which enhanced IL-6 production in the absence of cAMP accumulation. Two other peptides which are known to activate pituitary adenylate cyclase, corticotropin-releasing factor and GRF failed to stimulate IL-6 production in pituitary cells. Using discontinuous Percoll gradients to fractionate the pituitary cells, the greatest PACAP38-stimulated IL-6 secretion was observed in the low density fraction 1 (F1). This fraction also contained the highest percentage of folliculo-stellate (FS) cells, one of the nonhormone secreting pituitary cells. However, the largest PACAP38-induced accumulation of cAMP was observed in F4. These results suggest that the production of IL-6 stimulated by PACAP and CGRP is mediated by the adenylate cyclase/protein kinase A signal transduction system. FS cells appear to be the most likely target cell type for PACAP-induced IL-6 production. However, IL-6 producing FS cells may not be an exclusive target for PACAP in the pituitary.
The high and low affinity binding sites for PACAP were identified in rat astrocytes using [125I]PACAP27 as the labeled ligand. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites, with the dissociation constant (Kd) = 1.22 +/- 0.4 nM, the binding maximal capacity (Bmax) = 821 +/- 218 fmols/mg protein for the high affinity binding site, and Kd = 0.59 +/- 0.06 microM, Bmax = 563 +/- 12 pmols/mg protein for the low affinity binding site, respectively. The specificity of [125I]PACAP27 binding was tested using PACAP38 and peptides structurally related to PACAP, such as VIP, GHRF, PHI, secretin and glucagon. PACAP38 completely displaced the binding of [125I]PACAP27 and Scatchard analysis also indicated the presence of two classes of binding sites with similar Kd and Bmax to those for PACAP27. VIP and GHRF competed with [125I]PACAP27, but to a much lesser extent than unlabeled PACAP27 in binding. Other peptides tested did not displace the binding of [125I]PACAP27 at 10(-6) M.
Leukotriene B4 (LTB4), one of 5-lipoxygenase (5-LO) products of arachidonic acid, was reported to be more potent than leukotriene B5 (LTB5), one of 5-LO products of eicosapentaenoic acid, in the activation of neutrophil functions through the differential potency between these leukotrienes in calcium mobilization. So we compared the effect of LTB4 and LTB5 on the induction of interleukin 1 (IL-1)-like activity and calcium mobilization in human blood monocytes. LTB4 significantly augmented IL-1-like activity in monocytes, however, LTB5 had no significant effect. Also LTB4 was apparently more potent than LTB5 in inducing calcium mobilization in fura-2-loaded monocytes. IL-1 production in monocytes was reported to be partly dependent on cytosolic free calcium. These results, therefore, may indicate that the different activity between LTB4 and LTB5 in the enhancement of IL-1-like activity could be partly ascribed to the different potency in inducing calcium mobilization between LTB4 and LTB5 in human blood monocytes.