Biomedical subjects

I Tatsuno

Publications and source records attributed to I Tatsuno.

At least 19 recordsLinked to original sources

PubMed1992

Pituitary adenylate cyclase activating polypeptide and vasoactive intestinal peptide increase cytosolic free calcium concentration in cultured rat hippocampal neurons.

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)

Animals↗

PubMed1991

Role of 5-lipoxygenase products of arachidonic acid in cell-to-cell interaction between macrophages and natural killer cells in rat spleen.

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.

Animals↗

PubMed1991

Hypothalamic binding sites for pituitary adenylate cyclase activating polypeptide: characterization and molecular identification.

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.

Adenylyl Cyclases↗

PubMed1991

Glucocorticoid upregulation of interleukin 1 receptor expression in a glioblastoma cell line.

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.

Cell Line↗

PubMed1991

Inhibition of mitogen-stimulated proliferation of murine splenocytes by a novel neuropeptide, pituitary adenylate cyclase activating polypeptide: a comparative study with vasoactive intestinal peptide.

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.

Animals↗

PubMed1991

Neuropeptide regulation of interleukin-6 production from the pituitary: stimulation by pituitary adenylate cyclase activating polypeptide and calcitonin gene-related peptide.

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.

Animals↗

PubMed1990

Demonstration of specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytes.

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.

Analysis of Variance↗

PubMed1990

Comparison of the effect between leukotriene B4 and leukotriene B5 on the induction of interleukin 1-like activity and calcium mobilizing activity in human blood monocytes.

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.

Calcium↗

PubMed1990

Characterization and distribution of binding sites for the hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide.

A novel bioactive peptide was recently isolated from ovine hypothalamus and was named PACAP (pituitary adenylate cyclase-activating polypeptide). PACAP was present in two bioactive, amidated forms, PACAP27 and PACAP38 (27 and 38 amino acids, respectively), and showed a 68% sequence homology with vasoactive intestinal peptide (VIP) in the N-terminal 28 residues. PACAP38 was at least 1000 times more potent than VIP in stimulating adenylate cyclase in pituitary cells, but both peptides exhibited comparable vasodepressor activity. Thus, we sought to determine whether PACAP acts on specific binding sites in the anterior pituitary or other tissues and whether these binding sites are different from those of VIP. Binding of [125I] PACAP27 to freshly prepared rat anterior pituitary membranes in the presence and absence of 212 nM unlabeled PACAP27 was specific, saturable, and more rapid at 22 C than at 4 C. Scatchard analysis of this binding site using increasing doses of unlabeled PACAP27 revealed a single high affinity site with a Kd of 446 +/- 141 pM and a maximum number of sites of 1312 +/- 182 fmol/mg protein. These results do not exclude the possibility of a second pituitary binding site with significantly lower affinity. Unlabeled PACAP38 and PACAP38OH exhibited significantly higher affinity binding (3- to 5-fold) than PACAP27 with a similar number of pituitary sites. A variable distribution of binding sites was observed between PACAP27 and VIP when binding to different tissue membranes was measured with 125I-labeled peptides. Very high specific binding of both PACAP27 and VIP was observed in lung membranes. An almost identical relative magnitude of binding was observed between PACAP27 and VIP in lung, liver, duodenum, ovary, and thymus. However, whereas PACAP27 binding to hypothalamic and pituitary membranes was great, VIP binding to these tissues was almost absent. To determine if VIP and PACAP might share a binding site in peripheral tissues, displacement curves were generated using [125I]PACAP27 binding to lung membranes and VIP, PACAP27, and PACAP38 as unlabeled ligands. VIP was highly potent in displacing [125I] PACAP27 binding in lung membrane, and the IC50 values for all three of these peptides were between 1-10 nM. These results suggest that 1) a saturable, high affinity binding site for PACAP is present on anterior pituitary membranes; 2) PACAP27 and PACAP38, but not VIP, share this binding site in the anterior pituitary and possibly the hypothalamus; and 3) PACAP27, PACAP38, and VIP share a similar or identical binding site on lung membranes and possibly other peripheral tissues.

Animals↗

PubMed1989

Comparison of the effect of lipoxygenase metabolites of arachidonic acid and eicosapentaenoic acid on human natural killer cell cytotoxicity.

We compared in vitro effect of lipoxygenase (LO) products derived from arachidonic acid (AA) and eicosapentaenoic acid (EPA) on cytotoxic activity of human natural killer (NK) cell against human erythroleukemia cell line K-562. Leukotriene B4 (LTB4) derived from AA was found to significantly augment NK cell activity compared to the control level (in the absence of LTB). LTB5 showed a weak, but not significant, enhancing effect on NK cell activity. LTB4 was significantly more potent than LTB5 in the enhancement of NK cell activity. On the other hand, both 5- and 15-hydroperoxy fatty acids derived from AA and EPA significantly enhanced NK cell activity compared to the control level with similar potencies.

Arachidonic Acids↗

PubMed1988