Signaling by the Escherichia coli aspartate chemoreceptor Tar with a single cytoplasmic domain per dimer.

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.

Geranylgeranyl-pyrophosphate, an isoprenoid of mevalonate cascade, is a critical compound for rat primary cultured cortical neurons to protect the cell death induced by 3-hydroxy-3-methylglutaryl-CoA reductase inhibition.

We investigated the role of the intrinsic mevalonate cascade in the neuronal cell death (NCD) induced by the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in rat primary cortical neurons cultured from the brains of 17-d-old fetal SD rats. HMG-CoA reductase inhibitors induced NCD [HMG-CoA reductase inhibitor-induced NCD (H-NCD)] in time- and dose-dependent manners. The apoptotic characteristics were revealed by the formation of the DNA ladder and by the electron microscopical observation. During the progression of H-NCD, p53 was induced followed by the expression of Bax. Although the mevalonate completely inhibited H-NCD, the cholesterol did not. Thus, we examined two major metabolites of mevalonate, geranylgeranyl-pyrophosphate (GGPP) and farnesyl-pyrophosphate (FPP), using a novel liposome system for uptake into the cells. GGPP, not FPP, prohibited H-NCD with inhibition of the induction of p53 and Bax. The inhibition of HMG-CoA reductase decreased the amount of membrane-associated Rho small GTPase families, but not Ras small GTPase, and GGPP restored the blockage by HMG-CoA reductase inhibitor in the translocation or redistribution of Rho small GTPase families to membrane. These data indicated that (1) the inhibition of the intrinsic mevalonate cascade induces the apoptotic NCD with the induction of p53 followed by that of Bax, (2) the inhibition of HMG-CoA reductase concomitantly causes blockage of the translocation or redistribution of Rho small GTPase families, not Ras small GTPase, to membrane, and (3) GGPP, not FPP, is one of the essential metabolites in the mevalonate cascade for protecting neurons from H-NCD.

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.

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.

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.

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)

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.

Complete nucleotide sequence of the prophage VT1-Sakai carrying the Shiga toxin 1 genes of the enterohemorrhagic Escherichia coli O157:H7 strain derived from the Sakai outbreak.

Shiga toxins 1 and 2 (Stx1 and Stx2) are encoded by prophages lysogenized in enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains. Lytic growth of the phage particles carrying the stx1 genes (stx1A and stx1B) of the EHEC O157:H7 strain RIMD 0509952, which was derived from the Sakai outbreak in 1996 in Japan, was induced after treatment with mitomycin C, but the plaque formation of the phage was not detected. We have determined the complete nucleotide sequence of the prophage VT1-Sakai. The integration site of the prophage was identified within the yehV gene at 47.7 min on the chromosome. The stx1 genes were downstream of the Q gene in the prophage genome, suggesting that their expression was regulated by the Q protein, the regulator of the late gene expression of the phage, which is similar to that of the stx1 or stx2 genes carried by the lambdoid phages reported previously. The sequences of the N gene and its recognition sites, nutL and nutR, were not homologous to those of the phages carrying the stx genes thus far reported, but they were very similar to those of bacteriophage phi21. The sequences of the repressor proteins, CI and Cro, that regulate expression of the early genes had low similarities with those of the known repressors of other phages, and their operator sequences were different from any sequence reported. These data suggest that multiple genetic recombination among bacteriophages with different immunities took place to generate the prophage VT1-Sakai. Comparison between the sequences of VT1-Sakai and lambda suggests that the ancestor of VT1-Sakai was produced by illegitimate excision, like lambda gal and bio phages.

Involvement of PACAP receptor in primary afferent fibre-evoked responses of ventral roots in the neonatal rat spinal cord.

The role of PACAP receptor in nociceptive transmission was investigated in vitro using maxadilan, a PACAP receptor selective agonist and max.d.4, a PACAP receptor selective antagonist. Potentials, from a ventral root (L3 - L5) of an isolated spinal cord preparation or a spinal cord - saphenous nerve - skin preparation from 0 - 3-day-old rats, were recorded extracellularly. In the isolated spinal cord preparation, single shock stimulation of a dorsal root at C-fibre strength induced a slow depolarizing response lasting about 30 s (slow ventral root potential; slow VRP) in the ipsilateral ventral root of the same segment. Bath-application of max. d.4 (0.01 - 3 microM) inhibited the slow VRP in a concentration-dependent manner. In the spinal cord - saphenous nerve - skin preparation, application of capsaicin (0.1 microM) to the skin evoked a depolarization of the ventral root. This response was also depressed by max.d.4 (1 microM). Application of maxadilan evoked a long-lasting depolarization in a concentration-dependent manner in the spinal cord preparation. In the presence of max.d.4 (0.3 microM), the concentration response curve of maxadilan was shifted to the right. Reverse transcription-polymerase chain reaction (RT - PCR) experiments demonstrated the existence of PACAP receptor and VPAC(2) receptor in the neonatal rat spinal cord and [(125)I]-PACAP27 binding was displaced almost completely by maxadilan and max.d.4, but not by vasoactive intestinal peptide (VIP). These data indicate that PACAP receptor is dominantly distributed in the neonatal rat spinal cord. The present study suggests that PACAP receptor may play an excitatory role in nociceptive transmission in the neonatal rat spinal cord.

Maxadilan is a specific agonist and its deleted peptide (M65) is a specific antagonist for PACAP type 1 receptor.

Maxadilan is a potent vasodilator peptide isolated from salivary glands extracts of the hematophagous sand fly. Recently, it was demonstrated that maxadilan binds to PACAP receptor type 1 in mammals, although maxadilan has no significant amino acid sequence homology with PACAP. In the present study, we demonstrated that maxadilan is a specific agonist of PACAP type 1 receptor (PACAP/VIP receptor 1; PVR1) as determined by the binding assay of [125I]PACAP27 and cAMP accumulation using CHO cells stably expressing PVR1, VIP1 receptor (PVR2), and VIP2 receptor (PVR3), and that the deleted peptide (#25-41) of maxadilan (termed as M65) is a specific antagonist of PVR1. In addition, maxadilan shares the binding sites for PACAP and stimulates cAMP in cultured rat cortical neurons. VIP stimulates cAMP accumulation probably through the binding to PVR1 since M65 blocks the VIP-induced cAMP accumulation in cultured rat cortical neurons.

Specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat cultured astrocytes: molecular identification and interaction with vasoactive intestinal peptide (VIP).

Molecular identification of the binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) and the effect of vasoactive intestinal peptide (VIP) on the specific binding sites for PACAP in rat cultured astrocyte membrane preparations were investigated. Affinity cross-linking of astrocyte membrane preparations with [125I]PACAP27 showed the presence of a 60 kDa radiolabeled ligand-receptor complex. The labeling of this band was completely abolished in the presence of 10(-8) M or higher concentrations of unlabeled PACAP27. The molecular weight of this binding protein was estimated to be 57 kDa assuming an equimolar interaction of ligand and receptor in the 60 kDa complex. The labeling of [125I]PACAP27 binding to this binding protein was partly reduced by the addition of 10(-6) M VIP, but not by 10(-8) M. In the binding assay, VIP displaced the specific binding of [125I]PACAP27 at 10(-7) M or a greater concentration. Displacement of [125I]PACAP27 binding by unlabeled PACAP27 was analyzed in the presence or absence of 10(-6) M VIP. VIP at 10(-6) M reduced the maximal binding capacity (Bmax) of the high affinity binding site for PACAP27 by about 50% but did not alter the Bmax of the low affinity binding site. The dissociation constants (Kd) for both the high and low affinity binding sites were unaltered. These results indicate that PACAP binds to a 57 kDa membrane protein with high affinity and that VIP, at much higher concentrations, binds to this same binding site, suggesting that VIP mimics the biological action of PACAP in astrocytes at high concentrations.

Developmental changes of pituitary adenylate cyclase activating polypeptide (PACAP) and its receptor in the rat brain.

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.

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.

Pituitary adenylate cyclase-activating polypeptide (PACAP) mobilizes intracellular free calcium in cultured rat type-2, but not type-1, astrocytes.

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.

Regulation of interleukin-6 (IL-6) secretion in primary cultured rat astrocytes: synergism of interleukin-1 (IL-1) and pituitary adenylate cyclase activating polypeptide (PACAP).

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.

Pituitary adenylate cyclase-activating polypeptide protects rat-cultured cortical neurons from glutamate-induced cytotoxicity.

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.

Maxadilan specifically interacts with PAC1 receptor, which is a dominant form of PACAP/VIP family receptors in cultured rat cortical neurons.

Maxadilan is a potent vasodilator peptide isolated from salivary gland extracts of the hematophagous sand fly. Recently, the possibility was demonstrated that maxadilan binds to PAC1 receptor (PACAP, pituitary adenylate cyclase activating polypeptide type I receptor) in mammals. In the present study, we demonstrated that: (1) maxadilan specifically binds to PAC1 receptor and stimulates cyclic AMP accumulation in a dose-dependent manner in CHO cells stably expressing PAC1 receptor, not VIP (vasoactive intestinal polypeptide) receptors; that (2) the deleted peptide (amino acid #24-42) of maxadilan (termed max.d.4) also specifically binds to PAC1 receptor although max.d.4 inhibits cyclic AMP accumulation stimulated by both maxadilan and PACAP; and that (3) max.d.4 completely blocks the cyclic AMP accumulation induced by VIP in cultured rat cortical neurons. The expression of specific PACAP receptors in cultured rat cortical neurons was further investigated by the reverse transcription-polymerase chain reaction technique, which showed the presence of mRNA coding for PAC1 receptor among PACAP/VIP family receptors. These data indicate that maxadilan and max.d.4 represent important tools for clarifying the physiological role of PAC1 receptor, and that PAC1 receptor plays an important role in the regulation of the functions induced by PACAP in rat cultured cortical neurons.

Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates mitogen-activated protein kinase (MAPK) in cultured rat astrocytes.

Astrocytes, a subtype of glial cells, have been demonstrated to have an abundant number of receptors for pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide of the VIP/secretin family which stimulates cAMP accumulation 1000 times more potent than VIP in astrocytes. PACAP is reported to stimulate the proliferation of astrocytes at low concentrations at which it does not yet stimulate the cAMP accumulation. In the present study, we examined the effect of PACAP on the activation of mitogen-activated protein kinase (MAPK), one of the important intracellular signals for the proliferation, and compared it with that of epidermal growth factor (EGF). To investigate the activation of MAPK, we focused on ERK2, one of MAPK, in cultured rat astrocytes. The activation of ERK2 was determined by immunoblotting and measurement of the activity in terms of the phosphorylating activity of immunoprecipitates with MAPK antibody on myelin basic protein. One pM of PACAP38 temporarily activated ERK2 at 10 min. In contrast, EGF activated ERK2 from 10 min to 60 min continuously. As for the dose-response effect, PACAP stimulated ERK2 at as low a concentration as 10-14 M and peaked at 10-12 M. Thereafter, its activating effect gradually decreased at 10-10 M and returned to the basal level at 10-8 M, forming a bell-shaped dose-dependency. Neither an inhibitor of PKA (H89) nor inhibitors of PKC (staurosporine and calphostin C) had any effect on the ERK2 activation induced by 1 pM PACAP38. Dibutyryl cAMP suppressed ERK2 activity in a dose-dependent manner. These data clearly demonstrated that PACAP stimulates MAPK in both a PKA- and a PKC-independent manner in cultured rat astrocytes.

Geranylgeranylpyrophosphate plays a key role for the G1 to S transition in vascular smooth muscle cells.

Pravastatin, a HMG-CoA reductase inhibitor was found to inhibit DNA synthesis of vascular smooth muscle cells (VSMC) in a dose-dependent manner. Flow cytometric analysis demonstrated that pravastatin induced G1 arrest. Mevalonate restored the inhibitory effect of pravastatin on DNA synthesis and on cell cycle progression, suggesting the importance of mevalonate itself and/or its metabolites in VSMC proliferation. The major intermediate metabolites of mevalonate, geranylgeranyl-pyrophosphate (GGPP), farnesyl pyrophosphate (FPP) and IPP (isopentenyl pyrophosphate) were prepared in the form of liposomes, and the effects of GGPP, FPP and IPP on pravastatin induced inhibition of VSMC proliferation and G1 arrest were examined. Only GGPP restored the pravastatin-induced inhibition of DNA synthesis and G1 arrest. Pravastatin inhibited translocation of Rho small GTPase from cytosol to membrane. By the addition of GGPP, Rho small GTPase are geranylgeranylated and translocated to membranes during G1/S transition. These data suggest that GGPP, rather than FPP or IPP, is an essential metabolite among mevalonic acid metabolites for VSMC proliferation and the G1/S transition.

Antinociceptive effect of intrathecally administered pituitary adenylate cyclase activating polypeptide (PACAP) on the rat formalin test.

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.