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Introduction: Insulin-like growth factor (IGF)1 and IGF2 have neuroprotective effects, but less is known regarding how other members of the IGF system, including IGF binding proteins (IGFBPs) and the regulatory proteinase pappalysin-1 (PAPP-A) and its endogenous inhibitor stanniocalcin-2 (STC2) participate in this process. Here, we analyzed whether these members of the IGF system are modified in neurons and astrocytes in response to palmitic acid (PA), a fatty acid that induces cell stress when increased centrally., Methods: Primary hypothalamic astrocyte cultures from male and female PND2 rats and the pro-opiomelanocortin (POMC) neuronal cell line, mHypoA-POMC/GFP-2, were treated with PA, IGF1 or both. To analyze the role of STC2 in astrocytes, siRNA assays were employed., Results: In astrocytes of both sexes, PA rapidly increased cell stress factors followed by increased Pappa and Stc2 mRNA levels and then a decrease in Igf1, Igf2, and Igfbp2 expression and cell number. Exogenous IGF1 did not revert these effects. In mHypoA-POMC/GFP-2 neurons, PA reduced cell number and Pomc and Igf1 mRNA levels, and increased Igfbp2 and Stc2, again with no effect of exogenous IGF1. PA increased STC2 expression, but no effects of decreasing its levels by interference assays or exogenous STC2 treatment in astrocytes were found., Conclusions: The response of the IGF system to PA was cell and sex specific, but no protective effects of the IGFs were found. However, the modifications in hypothalamic PAPP-A and STC2 indicate that further studies are required to determine their role in the response to fatty acids and possibly in metabolic control., (© 2024 S. Karger AG, Basel.)

DNA damage is a central contributor to the aging process. In the brain, a major threat to the DNA is the considerable amount of reactive oxygen species produced, which can inflict oxidative DNA damage. This type of damage is removed by the base excision repair (BER) pathway, an essential DNA repair mechanism, which contributes to genome stability in the brain. Despite the crucial role of the BER pathway, insights into how this pathway is affected by aging in the human brain and the underlying regulatory mechanisms are very limited. By microarray analysis of four cortical brain regions from humans aged 20-99 years (n = 57), we show that the expression of core BER genes is largely downregulated during aging across brain regions. Moreover, we find that expression of many BER genes correlates positively with the expression of the neurotrophin brain-derived neurotrophic factor (BDNF) in the human brain. In line with this, we identify binding sites for the BDNF-activated transcription factor, cyclic-AMP response element-binding protein (CREB), in the promoter of most BER genes and confirm the ability of BDNF to regulate several BER genes by BDNF treatment of mouse primary hippocampal neurons. Together, these findings uncover the transcriptional landscape of BER genes during aging of the brain and suggest BDNF as an important regulator of BER in the human brain., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Normal brain development depends on tight temporal and spatial regulation of connections between cells. Mutations in L1cam, a member of the immunoglobulin (Ig) superfamily that mediate cell-cell contacts through homo- and heterophilic interactions, are associated with several developmental abnormalities of the nervous system, including mental retardation, limb spasticity, hydrocephalus, and corpus callosum aplasia. L1cam has been reported to be shed from the cell surface, but the significance of this during different phases of brain development is unknown. We here show that ADAM10-mediated shedding of L1cam is regulated by its fibronectin type III (FNIII) domains. Specifically, the third FNIII domain is important for maintaining a conformation where access to a membrane proximal cleavage site is restricted. To define the role of ADAM10/17/BACE1-mediated shedding of L1cam during brain development, we used a zebrafish model system. Knockdown of the zebrafish, l1camb, caused hydrocephalus, defects in axonal outgrowth, and myelination abnormalities. Rescue experiments with proteinase-resistant and soluble L1cam variants showed that proteolytic cleavage is not required for normal axonal outgrowth and development of the ventricular system. In contrast, metalloproteinase-mediated shedding is required for efficient myelination, and only specific fragments are able to mediate this stimulatory function of the shedded L1cam.

Formation of functional myelin sheaths within the central nervous system depends on expression of myelin basic protein (MBP). Following process extension and wrapping around axonal segments, this highly basic protein is required for compaction of the multi-layered membrane sheath produced by oligodendrocytes. MBP is hypothesized to be targeted to the membrane sheath by mRNA transport and local translation, which ensures that its expression is temporally and spatially restricted. The mechanistic details of how this might be regulated are still largely unknown, in particular because a model system that allows this process to be studied in vivo is lacking. We here show that the expression of the zebrafish MBP orthologs, mbpa and mbpb , is developmentally regulated, and that expression of specific mbpa isoforms is restricted to the peripheral nervous system. By analysis of transgenic zebrafish, which express a fluorescent reporter protein specifically in myelinating oligodendrocytes, we demonstrate that both mbpa and mbpb include a 3'UTR sequence, by which mRNA transport and translation is regulated in vivo . Further functional analysis suggests that: (1) the 3'UTRs delay the onset of protein expression; and that (2) several regulatory elements contribute to targeting of the mbp mRNA to the myelin sheath. Finally, we show that a pharmacological compound known to enhance neuronal activity stimulates the translation of Mbp in zebrafish in a 3'UTR-dependent manner. A similar effect was obtained following stimulation with a TrkB receptor agonist, and cell-based assays further confirmed that the receptor ligand, BDNF, in combination with other signals reversed the inhibitory effect of the 3'UTR on translation.

During development of the central nervous system not all axons are myelinated, and axons may have distinct myelination patterns. Furthermore, the number of myelin sheaths formed by each oligodendrocyte is highly variable. However, our current knowledge about the axo-glia communication that regulates the formation of myelin sheaths spatially and temporally is limited. By using axon-mimicking microfibers and a zebrafish model system, we show that axonal ephrin-A1 inhibits myelination. Ephrin-A1 interacts with EphA4 to activate the ephexin1-RhoA-Rock-myosin 2 signaling cascade and causes inhibition of oligodendrocyte process extension. Both in myelinating co-cultures and in zebrafish larvae, activation of EphA4 decreases myelination, whereas myelination is increased by inhibition of EphA4 signaling at different levels of the pathway, or by receptor knockdown. Mechanistically, the enhanced myelination is a result of a higher number of myelin sheaths formed by each oligodendrocyte, not an increased number of mature cells. Thus, we have identified EphA4 and ephrin-A1 as novel negative regulators of myelination. Our data suggest that activation of an EphA4-RhoA pathway in oligodendrocytes by axonal ephrin-A1 inhibits stable axo-glia interaction required for generating a myelin sheath., (© 2018 Wiley Periodicals, Inc.)

In the central nervous system, myelination of axons is required to ensure fast saltatory conduction and for survival of neurons. However, not all axons are myelinated, and the molecular mechanisms involved in guiding the oligodendrocyte processes toward the axons to be myelinated are not well understood. Only a few negative or positive guidance clues that are involved in regulating axo-glia interaction prior to myelination have been identified. One example is laminin, known to be required for early axo-glia interaction, which functions through α6β1 integrin. Here, we identify the Eph-ephrin family of guidance receptors as novel regulators of the initial axo-glia interaction, preceding myelination. We demonstrate that so-called forward and reverse signaling, mediated by members of both Eph and ephrin subfamilies, has distinct and opposing effects on processes extension and myelin sheet formation. EphA forward signaling inhibits oligodendrocyte process extension and myelin sheet formation, and blocking of bidirectional signaling through this receptor enhances myelination. Similarly, EphB forward signaling also reduces myelin membrane formation, but in contrast to EphA forward signaling, this occurs in an integrin-dependent manner, which can be reversed by overexpression of a constitutive active β1-integrin. Furthermore, ephrin-B reverse signaling induced by EphA4 or EphB1 enhances myelin sheet formation. Combined, this suggests that the Eph-ephrin receptors are important mediators of bidirectional signaling between axons and oligodendrocytes. It further implies that balancing Eph-ephrin forward and reverse signaling is important in the selection process of axons to be myelinated., (© The Author(s) 2015.)

Stanniocalcin-1 (STC1) is a disulfide-bound homodimeric glycoprotein, first identified as a hypocalcemic hormone important for maintaining calcium homeostasis in teleost fish. STC1 was later found to be widely expressed in mammals, although it is not believed to function in systemic calcium regulation in these species. Several physiological functions of STC1 have been reported, although many molecular details are still lacking. We here demonstrate that STC1 is an inhibitor of the metzincin metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A), which modulates insulin-like growth factor (IGF) signaling through proteolytic cleavage of IGF-binding proteins (IGFBPs). STC1 potently (Ki = 68 pm) inhibits PAPP-A cleavage of IGFBP-4, and we show in a cell-based assay that STC1 effectively antagonizes PAPP-A-mediated type 1 IGF receptor (IGF1R) phosphorylation. It has recently been found that the homologous STC2 inhibits PAPP-A proteolytic activity, and that this depends on the formation of a covalent complex between the inhibitor and the proteinase, mediated by Cys-120 of STC2. We find that STC1 is unable to bind covalently to PAPP-A, in agreement with the absence of a corresponding cysteine residue. It rather binds to PAPP-A with high affinity (KD = 75 pm). We further demonstrate that both STC1 and STC2 show inhibitory activity toward PAPP-A2, but not selected serine proteinases and metalloproteinases. We therefore conclude that the STCs are proteinase inhibitors, probably restricted in specificity to the pappalysin family of metzincin metalloproteinases. Our data are the first to identify STC1 as a proteinase inhibitor, suggesting a previously unrecognized function of STC1 in the IGF system., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mammalian stanniocalcin-2 (STC2) is a secreted polypeptide widely expressed in developing and adult tissues. However, although transgenic expression in mice is known to cause severe dwarfism, and targeted deletion of STC2 causes increased postnatal growth, its precise biological role is still unknown. We found that STC2 potently inhibits the proteolytic activity of the growth-promoting metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). Proteolytic inhibition requires covalent binding of STC2 to PAPP-A and is mediated by a disulfide bond, which involves Cys-120 of STC2. Binding of STC2 prevents PAPP-A cleavage of insulin-like growth factor-binding protein (IGFBP)-4 and hence release within tissues of bioactive IGF, required for normal growth. Concordantly, we show that STC2 efficiently inhibits PAPP-A-mediated IGF receptor signaling in vitro and that transgenic mice expressing a mutated variant of STC2, STC2(C120A), which is unable to inhibit PAPP-A, grow like wild-type mice. Our work identifies STC2 as a novel proteinase inhibitor and a previously unrecognized extracellular component of the IGF system., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Pregnancy-associated plasma protein A2 (PAPP-A2, also known as pappalysin-2) is a large metalloproteinase that is known to be required for normal postnatal growth and bone development in mice. We here report the detection of zebrafish papp-a2 mRNA in the chordamesoderm, notochord and lower jaw of zebrafish (Danio rerio) embryos, and that papp-a2-knockdown embryos display broadened axial mesoderm, notochord bends and severely reduced cranial cartilages. Genetic data link these phenotypes to insulin-like growth factor (Igf)-binding protein-3 (Igfbp-3) and bone morphogenetic protein (Bmp) signaling, and biochemical analysis show specific Igfbp-3 proteolysis by Papp-a2, implicating Papp-a2 in the modulation of Bmp signaling by Igfbp-3 proteolysis. Knockdown of papp-a2 additionally resulted in angiogenesis defects, strikingly similar to previous observations in embryos with mutations in components of the Notch system. Accordingly, we find that Notch signaling is modulated by Papp-a2 in vivo, and, furthermore, that human PAPP-A2 is capable of modulating Notch signaling independently of its proteolytic activity in cell culture. Based on these results, we conclude that Papp-a2 modulates Bmp and Notch signaling by independent mechanisms in zebrafish embryos. In conclusion, these data link pappalysin function in zebrafish to two different signaling pathways outside the IGF system., (© 2014. Published by The Company of Biologists Ltd.)

In the developing nervous system, abundant synthesis of myelin basic protein (MBP) in oligodendrocytes is required for the formation of compact myelin sheaths around axons. The MBP mRNA is known to be transported into the processes of oligodendrocytes. However, knowledge of the regulatory mechanisms that ensure the tight temporal and spatial control of MBP translation within these processes is limited. Here, we have identified novel regions within the 3'-UTR of the MBP mRNA that are responsible for the regulation of its translation, and we have demonstrated that each of the mRNA-binding proteins heterogeneous nuclear ribonucleoprotein (hnRNP)-A2, hnRNP-K and hnRNP-E1 serve distinct functions to regulate controlled and localized protein synthesis. hnRNP-A2 is responsible for mRNA transport, not for translational inhibition. By contrast, hnRNP-K and hnRNP-E1 play opposing roles in the translational regulation of MBP mRNA. We have identified shared binding sites within the 3'-UTR, and show that translation is promoted by the exchange of inhibitory hnRNP-E1 for stimulatory hnRNP-K. We further show that this molecular switch in the MBP messenger RNA-ribonucleoprotein (mRNP) complex, which regulates the synthesis of MBP, is important for the normal growth and extension of myelin sheets.

Background: Pregnancy-associated plasma protein-A (PAPP-A) is a local regulator of insulin-like growth factor (IGF) bioavailability in physiological systems, but many structural and functional aspects of the metzincin metalloproteinase remain to be elucidated. PAPP-A cleaves IGF binding protein (IGFBP)-4 and IGFBP-5. Cleavage of IGFBP-4, but not IGFBP-5, depends on the binding of IGF before proteolysis by PAPP-A can occur. The paralogue PAPP-A2 has two substrates among the six IGFBPs: IGFBP-3 and IGFBP-5., Methods: Sets of chimeric proteins between IGFBP-4 and -5, and IGFBP-3 and -5 were constructed to investigate the structural requirements for IGF modulation. At the proteinase level, we investigated the importance of individual acidic amino acids positioned in the proteolytic domain of PAPP-A for proteolytic activity against IGFBP-4 and -5. Interaction between PAPP-A and its substrates was analyzed by surface plasmon resonance., Results and Conclusion: We provide data suggesting that the C-terminal domain of the IGFBPs is responsible for IGF-dependent modulation of access to the scissile bond. Loss or reduction of IGFBP proteolysis by PAPP-A was observed upon mutation of residues positioned in the unique 63-residue stretch separating the zinc and Met-turn motifs, and in the short sequence following the Met-turn methionine. A model of the proteolytic domain of PAPP-A suggests the presence of structural calcium ions in the C-terminal subdomain, implicated in IGFBP substrate interactions., General Significance: Detailed knowledge of interactions between PAPP-A and its substrates is required to understand the modulatory role of PAPP-A on IGF receptor stimulation.

Myelination in the central nervous system provides a unique example of how cells establish asymmetry. The myelinating cell, the oligodendrocyte, extends processes to and wraps multiple axons of different diameter, keeping the number of wraps proportional to the axon diameter. Local regulation of protein synthesis represents one mechanism used to control the different requirements for myelin sheath at each axo-glia interaction. Prior work has established that β1-integrins are involved in the axoglial interactions that initiate myelination. Here, we show that integrin activation regulates translation of a key sheath protein, myelin basic protein (MBP), by reversing the inhibitory effect of the mRNA 3'UTR. During oligodendrocyte differentiation and myelination α6β1-integrin interacts with hnRNP-K, an mRNA-binding protein, which binds to MBP mRNA and translocates from the nucleus to the myelin sheath. Furthermore, knockdown of hnRNP-K inhibits MBP protein synthesis during myelination. Together, these results identify a novel pathway by which axoglial adhesion molecules coordinate MBP synthesis with myelin sheath formation.

The understanding of how adhesion molecules mediate the axon-glial interactions in the CNS that ensure target-dependent survival of oligodendrocytes and initiate myelination remains incomplete. Here, we investigate how signals from adhesion molecules can be integrated to regulate these initial steps of myelination. We first demonstrate that the Ig superfamily molecule contactin is associated in oligodendrocytes with integrins, extracellular matrix receptors that regulate target-dependent survival by amplification of growth factor signaling. This amplification is inhibited by small interfering RNA-mediated knockdown of contactin in oligodendrocytes. In contrast, the presence of L1-Fc, the extracellular portion of a contactin ligand expressed on axons, enhanced survival and additionally promoted myelination in cocultures of neurons and oligodendrocytes. We further demonstrate that the signals from contactin and integrin are integrated by differential phosphorylation of the Src family kinase Fyn. Integrin induced dephosphorylation of the inhibitory Tyr-531, whereas contactin increased phosphorylation of both Tyr-531 and the activating Tyr-420. The combined effect is an enhanced activity of Fyn and also a dynamic regulation of the phosphorylation/dephosphorylation balance of Fyn, as required for normal cell adhesion and spreading. We conclude, therefore, that a novel integrin/contactin complex coordinates signals from extracellular matrix and the axonal surface to regulate both oligodendrocyte survival and myelination by controlling Fyn activity.

Background: Gene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenability to genetic and experimental manipulation and because it possess a large number of duplicated genes., Methodology/principal Findings: We report the identification and characterization of two hypoxia-inducible genes in zebrafish that are co-ortholgs of human IGF binding protein-1 (IGFBP-1). IGFBP-1 is a secreted protein that binds to IGF and modulates IGF actions in somatic growth, development, and aging. Like their human and mouse counterparts, in adult zebrafish igfbp-1a and igfbp-1b are exclusively expressed in the liver. During embryogenesis, the two genes are expressed in overlapping spatial domains but with distinct temporal patterns. While zebrafish IGFBP-1a mRNA was easily detected throughout embryogenesis, IGFBP-1b mRNA was detectable only in advanced stages. Hypoxia induces igfbp-1a expression in early embryogenesis, but induces the igfbp-1b expression later in embryogenesis. Both IGFBP-1a and -b are capable of IGF binding, but IGFBP-1b has much lower affinities for IGF-I and -II because of greater dissociation rates. Overexpression of IGFBP-1a and -1b in zebrafish embryos caused significant decreases in growth and developmental rates. When tested in cultured zebrafish embryonic cells, IGFBP-1a and -1b both inhibited IGF-1-induced cell proliferation but the activity of IGFBP-1b was significantly weaker., Conclusions/significance: These results indicate subfunction partitioning of the duplicated IGFBP-1 genes at the levels of gene expression, physiological regulation, protein structure, and biological actions. The duplicated IGFBP-1 may provide additional flexibility in fine-tuning IGF signaling activities under hypoxia and other catabolic conditions.

The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves both insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4) and -5 at a single site in their central domain causing the release of bioactive IGF. Inhibition of IGF signaling is relevant in human disease, and several drugs in development target the IGF receptor. However, inhibition of PAPP-A activity may be a valuable alternative. We have generated monoclonal phage-derived single chain fragment variable (scFv) antibodies which selectively inhibit the cleavage of IGFBP-4 by PAPP-A, relevant under conditions where cleavage of IGFBP-4 represents the final step in the delivery of IGF to the IGF receptor. None of the antibodies inhibited the homologous proteinase PAPP-A2, which allowed mapping of antibody binding by means of chimeras between PAPP-A and PAPP-A2 to the C-terminal Lin12-Notch repeat module, separated from the proteolytic domain by almost 1000 amino acids. Hence, the antibodies define a substrate binding exosite that can be targeted for the selective inhibition of PAPP-A proteolytic activity against IGFBP-4. In addition, we show that the Lin12-Notch repeat module reversibly binds a calcium ion and that bound calcium is required for antibody binding, providing a strategy for the further development of selective inhibitory compounds. To our knowledge these data represent the first example of differential inhibition of cleavage of natural proteinase substrates by exosite targeting. Generally, exosite inhibitors are less likely to affect the activity of related proteolytic enzymes with similar active site environments. In the case of PAPP-A, selective inhibition of IGFBP-4 cleavage by interference with exosite binding is a further advantage, as the activity against other known or unknown PAPP-A substrates, whose cleavage may not depend on binding to the same exosite, is not targeted.

Myelination is necessary both for rapid salutatory conduction and the long-term survival of the axon. In the CNS the myelin sheath is formed by the oligodendrocytes. Each oligodendrocyte myelinates several axons and, as the number of wraps around each axon is determined precisely by the axon diameter, this requires a close, highly regulated interaction between the axons and each of the oligodendrocyte processes. Adhesion molecules are likely to play an important role in the bi-directional signalling between axon and oligodendrocyte that underlies this interaction. Here we review the current knowledge of the function of adhesion molecules in the different phases of oligodendrocyte differentiation and myelination, and discuss how the properties of these proteins defined by other cell biological systems indicates potential roles in oligodendrocytes. We show how the function of a number of different adhesion and cell-cell interaction molecules such as polysialic acid neural cell adhesion molecule, Lingo-1, Notch, neuregulin, integrins and extracellullar matrix proteins provide negative and positive signals that coordinate the formation of the myelin membrane. Compiling this information from a number of different cell biological and genetic experiments helps us to understand the pathology of multiple sclerosis and direct new areas of research that might eventually lead to potential drug targets to increase remyelination.

The biological activity of IGF-I and -II is controlled by six binding proteins (IGFBPs), preventing the IGFs from interacting with the IGF receptor. Proteolytic cleavage of IGFBPs is one mechanism by which IGF can be released to bind the receptor. The IGFBPs are usually studied individually, although the presence of more than one of the IGFBPs in most tissues suggests a cooperative function. Thus, the IGFBPs are part of regulatory networks with proteolytic enzymes in one end and the IGF receptor in the other end. We have established a model system that allows analysis of the dynamics between IGF, IGFBP-4 and -5, the IGF receptor, and the proteolytic enzyme PAPP-A, which specifically cleaves both IGFBP-4 and -5. We demonstrate different mechanisms of IGF release from IGFBP-4 and -5: cooperative binding to IGF is observed for the proteolytic fragments of IGFBP-5, but not fragments of IGFBP-4. Furthermore, we find that PAPP-A-mediated IGF-dependent cleavage of IGFBP-4 is inhibited by IGFBP-5, which sequesters IGF from IGFBP-4, and that cleavage of both IGFBP-4 and -5 is required for the release of bioactive IGF. Finally, we show that cell surface-localized proteolysis of IGFBP-4 represents the final regulatory step of efficient IGF delivery to the receptor. Our data define a regulatory system in which molar ratios between the IGFBPs and IGF and between the different IGFBPs, sequential proteolytic cleavage of the IGFBPs, and surface association of the activating proteinase are key elements in the regulation of IGF receptor stimulation.

The metzincin metalloproteinase pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1) promotes cell growth by proteolytic cleavage of insulin-like growth factor-binding proteins 4 and 5, causing the release of bound insulin-like growth factors. PAPP-A binds an unknown cell-surface heparan sulfate proteoglycan, suggesting that it controls insulin-like growth factor signaling spatially. In human pregnancy, the majority of PAPP-A circulates as a disulfide-bonded complex with its inhibitor, the proform of eosinophil major basic protein (proMBP). Interestingly, Ser-62 of proMBP is substituted with a glycosaminoglycan (GAG) chain, possibly a heparan sulfate type, and the PAPP-A.proMBP complex is unable to bind to the cell surface. We show here that proMBP detaches surface-bound PAPP-A in a process that depends on the proMBP GAG and also on the formation of intermolecular disulfide bonds between PAPP-A and proMBP. Unlike what was expected, we demonstrate that the GAG of proMBP is not required for PAPP-A.proMBP complex formation and that proMBP residues His-137, Ser-178, Arg-179, and Asn-181 are important for the recognition of PAPP-A. Using a mouse model, we find that the half-life of circulating PAPP-A and proMBP in complex is severalfold higher than both of the uncomplexed proteins, further suggesting that the PAPP-A.proMBP complex is formed at the cell surface in vivo rather than in the circulation. Further supporting this, we show that formation of the PAPP-A.proMBP complex at the cell surface proceeds rapidly compared with the slow rate of complex formation in solution. Because both PAPP-A and proMBP are expressed ubiquitously, this model may be applicable to many tissues in which insulin-like growth factor bioavailability is locally regulated.

Insulin-like growth factor (IGF)-I and -II function in normal physiology to control growth, development, and differentiation, but are also important in pathophysiological conditions, particularly in cancer. The biological effects of the IGFs are mediated by the IGF-I receptor (IGFR), a covalent homodimer composed of two alpha and two beta chains, similar in structure to the insulin receptor (IR). To allow measurement of the stimulation of IGFR in living cells, we developed an assay based on bioluminescence resonance energy transfer (BRET) between a donor molecule, Renilla luciferase, and an acceptor fluorophore, enhanced yellow fluorescent protein (EYFP). Initial attempts based on fusion of the luciferase to IGFR, and EYFP to IGFR, or to downstream signaling molecules, insulin receptor substrate-1 (IRS1) or protein tyrosine phosphatases-1B (PTP-1B), failed. However, similar experiments with IR, carried our in parallel, proved successful. We therefore, constructed assays based on chimeric IGFR/IR proteins, in which the ligand binding site was derived from IGFR. With the most efficient assay, in which the luciferase is fused to a chimeric receptor with the entire intracellular portion derived from IR, and EYFP fused to PTP-1B, IGF activity was measured specifically with sensitivity similar to the corresponding assay for insulin, based on IR. The established system allows efficient evaluation of candidate ligand- or receptor-directed molecules for the modulation of IGF activities. Furthermore, we demonstrate that a set of inhibitory IGF binding proteins (IGFBPs) or activating IGFBP-specific proteinases, unique to the IGF system, may serve as potential targets. In addition to screening, real-time measurement of IGFR stimulation may be important in efforts to understand the kinetics of receptor stimulation, in particular differences between IGFR and IR.

The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves a subset of insulin-like growth factor binding proteins (IGFBP), which inhibit the activities of insulin-like growth factor (IGF). Through this proteolytic activity, PAPP-A is believed to regulate IGF bioavailability in several biological systems, including the human reproductive system and the cardiovascular system. PAPP-A adheres to mammalian cells by interactions with glycosaminoglycan (GAG), thus targeting the proteolytic activity of PAPP-A to the cell surface. Based on site-directed mutagenesis, we here delineate the PAPP-A GAG-binding site in the C-terminal modules CCP3 and CCP4. Using heparin affinity chromatography, commonly employed in such studies, we define three clusters of arginines and lysines of CCP3, which are important for the interaction of PAPP-A with heparin. In a model of PAPP-A CCP3-CCP4, basic residues of these sequence clusters form a contiguous patch located on one side of the structure. Binding to the unknown, natural cell surface receptor of PAPP-A, assessed by flow cytometry, also depends on residues of these three basic clusters. However, single or double residue substitutions generally have a modest effect on PAPP-A heparin binding assessed by chromatography, but cell surface adhesion was critically reduced by several of these substitutions, emphasizing the relevance of analysis by flow cytometry. The contributions of positively charged residues located in CCP4 were all minor when analyzed by heparin affinity chromatography. However, the mutation of CCP4 residues Arg1459 and Lys1460 to Ala almost abrogated cell surface adhesion. Furthermore, when acidic residues of the homologous proteinase PAPP-A2 (Asp1547, Glu1555 and Glu1567) were introduced into the corresponding positions in the sequence of PAPP-A, located in each of the three basic clusters of CCP3, binding to heparin was strongly impaired and cell surface binding was abrogated. This explains, at least in part, why PAPP-A2 lacks the ability of cell surface adhesion, and further emphasizes the role of the basic clusters defined in PAPP-A.

The activities of insulin-like growth factor (IGF)-I and -II are regulated by IGF-binding proteins (IGFBPs). Cleavage of IGFBP-4 by the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) causes release of bound IGF and has been established in several biological systems including the human reproductive system. Using flow cytometry, we first demonstrate that PAPP-A reversibly binds to the cell surface of several cell types analyzed. Heparin and heparan sulfate, but not dermatan or chondroitin sulfate, effectively compete for PAPP-A surface binding, and because incubation of cells with heparinase abrogated PAPP-A adhesion, binding is probably mediated by a cell surface heparan sulfate proteoglycan. Furthermore, the proteolytic activity of PAPP-A is preserved while bound to cells, suggesting that adhesion functions to target its activity to the vicinity of the IGF receptor, decreasing the probability that released IGF is captured by another IGFBP molecule before receptor binding. This mechanism potentially functions in both autocrine and paracrine regulation, as PAPP-A need not be synthesized in a cell to which it adheres. A truncated PAPP-A variant without the five short consensus repeats in the C-terminal third of the 1547-residue PAPP-A subunit, lacked surface binding. We also show that PAPP-A2, a recently discovered IGFBP-5 proteinase with homology to PAPP-A, does not bind cells. This finding allowed further mapping of the PAPP-A adhesion site to short consensus repeat modules 3 and 4 by the expression and analysis of nine PAPP-A/PAPP-A2 chimeras. Interestingly, the proteolytically inactive, disulfide-bound complex of PAPP-A and the proform of eosinophil major basic protein (proMBP), PAPP-A.proMBP, shows only weak surface binding, probably because the adhesion site of PAPP-A is occupied by heparan sulfate, known to be covalently bound to proMBP. This hypothesis was further substantiated by demonstrating that heparinase treatment of PAPP-A.proMBP restores surface binding. We finally propose a model in which IGF bioactivity is regulated by reversible cell surface binding of PAPP-A, which in turn is regulated by proMBP.

Human pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor (IGF) binding protein-4 (IGFBP-4), causing a dramatic reduction in its affinity for IGF-I and -II. Through this mechanism, PAPP-A is a regulator of IGF bioactivity in several systems, including the human ovary and the cardiovascular system. PAPP-A belongs to the metzincin superfamily of zinc metalloproteinases, and is the founding member of a fifth metzincin family, the pappalysins. Herein, we first determined that PAPP-A cleaves IGFBP-4 at a single site (Met-135/Lys-136), and we analysed the influence of ionic strength, pH and zinc ion concentration on the cleavage reaction. Secondly, we sought to delineate the role of substrate residues in PAPP-A-mediated cleavage by the construction and analysis of 30 IGFBP-4 mutants in which various residues were replaced by alanine, by the analysis of eight mutants of IGFBP-5 (found recently to be a second PAPP-A substrate), and by cleavage analysis of synthetic peptides derived from IGFBP-4. Our data reveal a complex mode of substrate recognition and/or binding, pointing at important roles for several basic residues located up to 16 residues N-terminal to the scissile bond. An unexpected parallel can be drawn with an intracellular enzyme, the mitochondrial processing peptidase, that may help us to understand properties of the pappalysins. Further, proteinase-resistant variants of IGFBP-4 and -5, presented here, will be useful tools for the study of proteolysis in cell-based systems, and our finding that a synthetic peptide can be cleaved by PAPP-A provides the basis for development of quantitative assays for the investigation of PAPP-A enzyme kinetics.

Murine pregnancy-associated plasma protein-A (PAPP-A) cDNA encoding a 1545 amino-acid protein has been cloned. We have also identified and cloned cDNA that encodes a novel variant of PAPP-A, PAPP-Ai, carrying a 29-residue highly basic insert. The point of insertion corresponds to a junction between two exons in the human PAPP-A gene. The human intron flanked by these exons does not encode a homologous corresponding insert, which is unique to the mouse. The overall sequence identity between murine and human PAPP-A is 91%, and murine PAPP-A contains sequence motifs previously described in the sequence of human PAPP-A. Through expression in mammalian cells, we show that murine PAPP-A and PAPP-Ai are active metalloproteinases, both capable of cleaving insulin-like growth factor binding protein (IGFBP)-4 and -5. Cleavage of IGFBP-4 is dramatically enhanced by the addition of IGF, whereas cleavage of IGFBP-5 is slightly inhibited by IGF, as previously established with human PAPP-A. Surprisingly, however, quantitative analyses demonstrate that the murine PAPP-Ai cleaves IGFBP-4 very slowly compared to PAPP-A, even though its ability to cleave IGFBP-5 is unaffected by the presence of the insert. By RT-PCR analysis, we find that both variants are expressed in several tissues. The level of mRNA in the murine placenta does not exceed the levels of other tissues analyzed. Furthermore, the IGFBP-4-proteolytic activity of murine pregnancy serum is not elevated. This is in striking contrast to the increase seen in human pregnancy serum, and the expression of PAPP-A in the human placenta, which exceeds other tissues at least 250-fold. Interestingly, the position of the insert of PAPP-Ai, within the proteolytic domain, lies in close proximity to the cysteine residue, which in human PAPP-A forms a disulfide bond with the proform of eosinophil major basic protein (proMBP). ProMBP functions as a proteinase inhibitor in the PAPP-A-proMBP complex, but whether any mechanistic parallel on regulation of proteolytic activity can be drawn between the insert of PAPP-Ai and the linkage to proMBP is not known. Importantly, these data support the development of the mouse as a model organism for the study of PAPP-A, which must take into account the differences between the mouse and the human.

The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1-6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PAPP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482-His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.

Pregnancy-associated plasma protein-A (PAPP-A) has recently been identified as the proteinase responsible for cleavage of insulin-like growth factor binding protein (IGFBP)-4, an inhibitor of IGF action, in several biological fluids. Cleavage of IGFBP-4 by PAPP-A is believed to occur only in the presence of IGF. We here report that in addition to IGFBP-4, PAPP-A also cleaves IGFBP-5. Cleavage occurs at one site, between Ser-143 and Lys-144 of IGFBP-5. In the presence of IGF, IGFBP-4 and -5 are cleaved with similar rates by PAPP-A. Interestingly, cleavage of IGFBP-5 by PAPP-A does not require the presence of IGF, but is slightly inhibited by IGF. These findings have implications for the mechanism of proteolysis of IGFBP-4 by PAPP-A, suggesting that IGFBP-4 binds IGF, which then becomes a PAPP-A substrate. Using highly purified, recombinant proteins, we establish that (1) PAPP-A cleavage of IGFBP-4 can occur in the absence of IGF, although the rate of hydrolysis is very slow, and (2) IGF is unable to bind to PAPP-A. We thus conclude that IGF enhances proteolysis by binding to IGFBP-4, not by interaction with PAPP-A, which could not previously be ruled out.

A novel metalloproteinase with similarity to pregnancy-associated plasma protein-A (PAPP-A), which we denoted PAPP-A2, has been identified. Through expression in mammalian cells we showed that recombinant PAPP-A2 polypeptide of 1558 residues resulted from processing of a 1791-residue prepro-protein. Unlike PAPP-A, PAPP-A2 migrated as a monomer (of 220 kDa) in non-reducing SDS-polyacrylamide gel electrophoresis. The prepro-parts of PAPP-A2 and PAPP-A are not homologous, but mature PAPP-A2 shares 45% of its residues with PAPP-A. Because PAPP-A specifically cleaves insulin-like growth factor-binding protein (IGFBP)-4, one of six known modulators of IGF-I and -II, we looked for a possible PAPP-A2 substrate among the members of this family. We showed that PAPP-A2 specifically cleaved IGFBP-5 at one site, between Ser-143 and Lys-144. In contrast to the cleavage of IGFBP-4 by PAPP-A that strictly requires the presence of IGF, the cleavage of IGFBP-5 by PAPP-A2 was IGF-independent. Recent data firmly establish PAPP-A and IGFBP-4 as an important functional pair in several systems. Because of its close relationship with PAPP-A, both structurally and functionally, PAPP-A2 is a likely candidate IGFBP-5 proteinase in many tissues and conditioned media where IGFBP-5 proteolysis has been reported.

Pregnancy-associated plasma protein-A (PAPP-A), originally known from human pregnancy serum, has recently been demonstrated to be a metzincin superfamily metalloproteinase involved in normal and pathological insulin-like growth factor (IGF) physiology. PAPP-A specifically cleaves IGF-binding protein (IGFBP)-4, one of six antagonists of IGF action, which results in release of IGF bound to IGFBP-4. IGFBP-4 is the only known PAPP-A substrate. Its cleavage by PAPP-A uniquely depends on the presence of IGF. We here report mammalian expression and purification of recombinant 1547-residue PAPP-A (rPAPP-A). The recombinant protein is secreted as a homodimer of about 400 kDa composed of two 200-kDa disulfide-bound subunits. Antigenically and functionally, rPAPP-A behaves like the native protein. In human pregnancy, PAPP-A is known to circulate as a 500-kDa disulfide-bound 2:2 complex with the proform of eosinophil major basic protein (proMBP), PAPP-A/proMBP. A comparison between rPAPP-A and pregnancy serum PAPP-A/proMBP complex surprisingly reveals a difference greater than 100-fold in proteolytic activity, showing that proMBP functions as a proteinase inhibitor in vivo. We find that polyclonal antibodies against PAPP-A abrogate all detectable IGFBP-4 proteolytic activity in pregnancy serum, pointing at PAPP-A as the dominating, if not the only, IGFBP-4 proteinase present in the circulation. We further show that pregnancy serum and plasma contain traces (<1%) of uncomplexed PAPP-A with a much higher specific activity than the PAPP-A/proMBP complex. The measurable activity of the PAPP-A/proMBP complex probably results from the presence of a minor subpopulation of partly inhibited PAPP-A that exists in a 2:1 complex with proMBP. Inhibition of PAPP-A by proMBP represents a novel inhibitory mechanism with the enzyme irreversibly bound to its inhibitor by disulfide bonds.

5-Lipoxygenase products of arachidonic acid metabolism are thought to play a central role in the secondary amplification of the inflammatory response of several inflammatory diseases, including ulcerative colitis. FPL 64170XX is a selective inhibitor of the enzyme 5-lipoxygenase. Concentrations of leukotriene B4 and prostaglanding E2 in rectal dialysis fluid from 23 males with clinically and sigmoidoscopically active, distally located ulcerative colitis were measured by radioimmunoassays in a double-blind, placebo-controlled, parallel design study before and after rectal administration of an enema containing 0.5% of FPL 64170XX. Repeated measures analysis of leukotriene B4, after adjusting for baseline, showed a significant treatment effect (P = 0.0014). The concentration of leukotriene B4 from rectal dialysates in patients receiving the active drug dropped to 15% (95% confidence interval 5-40%) of the placebo level in the second dialysis following administration of FPL 64170XX 0.5%. By contrast, prostaglanding E2 concentrations doubled (P = 0.0068) in patients receiving FPL 64170XX 0.5% with no change in the placebo group. These findings demonstrate that a single dose of FPL 64170XX 0.5% enema selectively blocks the generation of the 5-lipoxygenase product, leukotriene B4, to a mean of 85% in the target tissue of inflammation. Topical administration of this new leukotriene synthesis inhibitor may prove to be a clinically useful approach to the treatment of active, distally located ulcerative colitis.

Background: Omeprazole is effective in the treatment of reflux oesophagitis, and it is important to determine the lower dose limit with still appropriate clinical efficacy., Methods: Patients with endoscopic oesophagitis grade 1-4 (N = 220) were randomized to double-blind treatment with 20 mg or 40 mg omeprazole daily for 4-8 weeks. Those healed after this initial treatment phase were re-randomized to double-blind treatment with 20 mg omeprazole daily (n = 67), 10 mg omeprazole daily (n = 68), or placebo (n = 33) for 6 months. Remission was defined as the absence of any endoscopic sign of oesophagitis., Results: Healing rates were increased with 40 mg omeprazole, the therapeutic gain compared with the 20-mg dose being 15% after 4 and 8 weeks. The proportion of patients in remission after 6 months was 59% with 20 mg omeprazole, 35% with 10 mg omeprazole, and 0% with placebo., Conclusion: Maintenance treatment with 10 mg omeprazole can prevent recurrence of oesophagitis in about one-third of patients with all grades of oesophagitis, and 20 mg omeprazole in about twice as many.

Background: 5-Lipoxygenase products of arachidonic acid metabolism are thought to play a central role in the secondary amplification of the inflammatory response in a number of human inflammatory diseases, such as ulcerative colitis. MK-0591 (3-(1((4-chlorophenyl)methyl)-3((1,1-dimethyl-ethyl)thio)-5(quinolin+ ++-2ylmethyl-oxy)-1H-indol-2yl)-2,2-dimethyl-propanoate) exerts its effect by binding to the 5-lipoxygenase activating protein, thereby inhibiting the translocation and activation of 5-lipoxygenase., Methods: Concentrations of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) in rectal dialysis fluid, ex vivo biosynthesis of LTB4 in whole blood, and urinary excretion of leukotriene E4 (LTE4) from 16 patients with mild to moderately active distally located ulcerative colitis were measured by use of radioimmunoassays in a double-blind, placebo-controlled parallel-design study before and after oral administration of a 250 mg dose of MK-0591 or placebo., Results: The mean LTB4 concentration in rectal dialysis fluid was lowered after MK-0591 by > 90% (p < 0.05) from 4 to 8 hours, with a maximum inhibition of 97.5% +/- 3.4% (mean +/- SD) at 20 to 24 hours after dosing, whereas PGE2 was unchanged. In whole blood, MK-0591 decreased ex vivo biosynthesis of LTB4 (p < 0.01), with a maximum inhibition of 96.4% +/- 2.1% at 4 hours after dosing. Urinary excretion of LTE4 was reduced by more than 85% (p < 0.001) from 4 to 48 hours. No adverse events were observed., Conclusion: These findings show that a single oral 250 mg dose of MK-0591 results in nearly complete blockade of systemic leukotriene production and LTB4 formation in the target tissue of inflammation (the rectum). Controlled multiple-dose trials to assess the clinical efficacy of this novel 5-lipoxygenase-activating protein inhibitor seem to be worthwhile.

Grade I oesophagitis is usually considered to be a less severe form of gastro-oesophageal reflux disease (GORD). However, with regard to symptom severity, patients without macroscopic mucosal lesions have been shown not to differ from those with more severe oesophagitis. A number of controlled trials on the efficacy of omeprazole in GORD have included patients with lower grades of the disease. The results show that the differences in efficacy between omeprazole and H2-receptor antagonists, which have been established for the treatment of erosive and ulcerative oesophagitis, also extend to patients with grade I oesophagitis (erythema and friability). In these studies, omeprazole provided more rapid symptom resolution and histological improvement than ranitidine. In one double-blind comparative trial, complete endoscopic normalization of the oesophageal mucosa was observed in 90% of patients with grade I oesophagitis within 4 weeks of treatment with omeprazole, 40 mg once daily, compared with 55% of those treated with ranitidine, 150 mg twice daily; at 8 weeks the mucosa in all patients in the omeprazole group had completely healed at endoscopy, while oesophagitis was still present in 21% of the patients receiving ranitidine. A separate 6-month, placebo-controlled maintenance study was performed in patients who had completed a short-term study and who had total relief from the major symptoms of GORD and complete healing of endoscopic oesophagitis. All patients given placebo had an endoscopic recurrence (i.e. endoscopic grade I or more) and this was associated with the return of symptoms in 75% of cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic inflammatory bowel disease (IBD) encompasses the disease entities, ulcerative colitis (UC) and Crohn's disease (CD). An aetiologic agent has not yet been defined and the diagnosis is based, therefore, on the sum of clinical, paraclinical, radiologic, endoscopic and histopathologic features. In recent years pathogenetic studies have focused on immune mechanisms, transmissible infectious agents, the potential role of the normal intestinal flora, dietary factors, enzymatic alterations and genetic features, in addition to vascular, neuromotor, allergic and psychologic factors. The prevalence of IBD is increased in first-degree relatives of patients and there is a high rate of disease concordance among monozygotic twins. Thus abnormal genes may encode for one or several immunoregulatory factors, while bacterial wall products seem to activate colonic inflammatory cells in a non-specific way, leading to increased production of cytokines, complement-derived peptides, eicosanoids, platelet activating factor, biogenic amines, kinins, chemotactic oligopeptides, and neuropeptides. The named soluble inflammatory mediators, in addition to free oxygen radicals, are considered responsible for the secondary amplification of the inflammatory process. The corner stones in medical therapy of IBD are still corticosteroids and sulphasalazine (SAZ). The new oral salicylates, which are analogues of SAZ or "slow release" preparations of 5-aminosalicylic acid (mesalazine), have provided a therapeutic progress, because they are tolerated better than SAZ. Immunosuppressive agents, such as azathioprine and 6-mercaptopurine, reduce the requirement for corticosteroids and are effective in refractory CD, but the effect is delayed up to several months. The therapeutic action of cyclosporine A is not sustained, but often associated with side effects. Metronidazole has a beneficial effect on perineal disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic inflammatory bowel disease (IBD) encompasses the disease entities, ulcerative colitis (UC) and Crohn's disease (CD). An aetiologic agent has not yet been defined and the diagnosis is based, therefore, on the sum of clinical, paraclinical, radiologic, endoscopic and histopathologic features. In recent years pathogenetic studies have focused on immune mechanisms, transmissible infectious agents, the potential role of the normal intestinal flora, dietary factors, enzymatic alterations and genetic features, in addition to vascular, neuromotor, allergic and psychologic factors. The corner stones in medical therapy of IBD are still corticosteroids and sulphasalazine (SAZ). The new oral salicylates, which are analogues of SAZ or "slow release" preparations of 5-aminosalicylic acid (mesalazine), have provided a therapeutic progress, because they are tolerated better than SAZ. Immunosuppressive agents, such as azathioprine and 6-mercaptopurine, reduce the requirement for corticosteroids and are effective in refractory CD, but the effect is delayed up to several months. The therapeutic action of cyclosporine A is not sustained, but often associated with side effects. Metronidazole has a beneficial effect on perineal disease. The efficacy of antimycobacterial drugs, sodium-cromoglycate, lidocaine, clonidine and sucralfate has been reported only in optimistic case stories and small open trials. A diet, rich in omega-3-fatty acids, modifies leukotriene (LT) production, but its clinical efficacy is insufficient. The first anti-leukotriene-drug, zileuton, has recently been evaluated and a significant, although insufficient, clinical response was obtained by a 70 per cent inhibition of rectal LTB4 synthesis. Dietary therapy may be useful as an adjunct to treatment of local complications in CD.(ABSTRACT TRUNCATED AT 250 WORDS)

The unique role of the enzyme 5-lipoxygenase (5-LO) in the production of leukotrienes (LTs) makes it a likely target for biochemical manipulation. The rationale for using 5-LO inhibitors for the treatment of inflammatory bowel disease (IBD) is based on the increased generation of LTs in the inflamed mucosa, LTB4 being the most potent chemotactic and chemokinetic metabolite of arachidonic acid. Furthermore, conventional drugs, such as corticosteroids, sulphasalazine, and 5-aminosalicylic acid, inhibit LT production and specific 5-LO inhibition accelerates healing in animal models of acute colitis. The compounds identified as 5-LO inhibitors can be divided into antioxidants, substrate-analogous, and a large miscellaneous group of inhibitors, where hydroxamic acids are potent and more selective inhibitors of 5-LO. The benzothiophene hydroxyurea, zileuton, is the first selective 5-LO inhibitor evaluated for the treatment of patients with IBD. An 800-mg oral dose of zileuton was shown to reduce LTB4, but not prostaglandin E2, concentrations by 75-85% in rectal dialysates from patients with active ulcerative colitis. The clinical efficacy of zileuton 800 mg b.i.d. has also been tested in a randomized, double-blind, placebo-controlled trial in similar patients. Zileuton significantly improved the symptom scores and the histology score, but not the sigmoidoscopy score, compared to pretreatment conditions and with response to placebo, the beneficial effects being most pronounced in patients not receiving concomitant sulphasalazine treatment. The mean inhibition of LTB4 in the target tissue of inflammation was 70%. The proof that any putative 5-LO inhibitor is blocking LT production is an important stage in assessing any such drug. The main disadvantage of existing new LT inhibitors relates to the high potency of LTs, and unless a higher level of inhibition can be achieved, endogenous LTs may still be present in sufficient amounts to produce their effects.

In a double-blind, parallel-group clinical trial of 195 patients with duodenal ulcers who after a short-term study had relief of pain and healed ulcers proved endoscopically, 65 were randomized to receive 20 mg omeprazole 3 days a week (once in the morning from Friday to Sunday), 64 to receive 10 mg omeprazole once daily in the morning, and 66 to receive placebo for up to 6 months. The patients underwent repeat endoscopy with biopsy of the gastric fundic mucosa (qualitative assessment of argyrophilic cell population), assessment of symptoms, and laboratory screening with measurement of basal serum gastrin concentrations at 3 and 6 months or more often if indicated by recurrence of symptoms. At 3 months, endoscopically proved ulcer relapse occurred in 16% receiving 20 mg omeprazole 3 days a week; 21% receiving 10 mg omeprazole daily; and 50% receiving placebo. At 6 months, corresponding rates were 23%, 27%, and 67% with 95% confidence intervals of difference between the placebo group and omeprazole groups of 28%-60% and 24%-56% (P less than 0.00001), respectively, and between omeprazole groups of -19%-11% (NS). No major clinical or laboratory side effects were noted. Thus both omeprazole regimens are effective and safe in preventing duodenal ulcer relapse.

Part I of this article, which appeared in the previous issue of the Journal, covered the following agents: histamine H2-receptor antagonists (cimetidine, ranitidine, famotidine, nizatidine); muscarinic-M1-receptor antagonists (pirenzepine); proton pump inhibitors (omeprazole); site-protective agents (colloidal bismuth subcitrate, sucralfate); antacids and prostaglandin analogues; antiemetics and prokinetics (metoclopramide, domperidone, cisapride); and antispasmodics. In Part II, we consider the anti-inflammatory salicylates, nonspecific antidiarrhoeal agents, laxatives and cathartics.

Leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) concentrations were measured in rectal dialysis fluid from ten patients with active ulcerative colitis before and after oral administration of 800 mg of the 5-lipoxygenase inhibitor A-64077. The median LTB4 level fell significantly, from 4.9 (range 0.6-20.4) ng/ml before treatment to 1.6 (0.3-5.7) ng/ml after 4 h and 0.7 (0.1-8.0) ng/ml after 8 h; it had returned to pretreatment levels by 28 h. The concentration of PGE2 did not change significantly. The increased generation of 5-lipoxygenase products, such as LTB4, in ulcerative colitis and the potent proinflammatory actions of these products suggest that they have an important role in the amplification of the inflammatory response. A controlled trial to assess the clinical efficacy of A-64077 seems worth while.

To establish whether concentrations of eicosanoids determined by equilibrium in vivo dialysis of faeces and equilibrium in vivo dialysis of rectum might predict a relapse in ulcerative colitis, 23 patients with completely inactive disease, maintained on sulphasalazine, stopped treatment and entered a prospective study. Concentrations of prostaglandin E2 were determined by radioimmunoassay on purified faecal and rectal dialysates at entry, at two weeks, and at two, six, and 12 months. If the above concentrations exceeded control concentrations (0.5 ng/ml and 1.0 ng/ml in faecal and rectal fluid, respectively) at any study day, the patient was allocated at random to double blind treatment with sulphasalazine 2 g/day, or placebo for six months. A relapse, defined as recurrence of symptoms accompanied by endoscopic inflammation occurred in none of six and in four of five patients allocated to sulphasalazine and placebo, respectively (p less than 0.05). In no case a normal rectal prostaglandin E2 concentration was associated with a relapse in the short term, but only two of 12 patients observed passively remained in remission. In retrospect, leukotriene B4 was a less sensitive predictor of relapse than prostaglandin E2. We conclude that raised concentrations of prostaglandin E2 in rectal dialysis fluid identify patients with a substantial risk of relapse.

To compare the local release of arachidonic acid metabolites in inflammatory diarrheal disease, in vivo equilibrium dialysis of the rectum was done in consecutive untreated patients with ulcerative colitis (n = 20), Crohn's colitis (n = 10), and Clostridium difficile colitis (n = 7). All patients had endoscopically proven rectal inflammation. Eicosanoid profiles were determined in rectal dialysates by radioimmunoassay after preliminary purification. Concentrations of prostaglandin E2, prostaglandin F2 alpha, and thromboxane B2, but not 6-keto-prostaglandin F1 alpha, were raised in all groups and compared with healthy controls. The highest levels within each group were obtained in patients with widespread epithelial damage, as judged by endoscopy. In patients with ulcerative colitis, an extreme rise in prostaglandin E2 and thromboxane B2 were observed. Similarly, concentrations of leukotriene B4 were substantially increased in ulcerative colitis, but in Crohn's colitis and Clostridium difficile colitis only those patients with rectal ulcerations showed elevations. These findings probably reflect more severe tissue damages in ulcerative colitis, but differences between disease groups in cell-to-cell interaction may also contribute. The data suggest, therefore, that therapeutic inhibition of lipoxygenase pathways may prove more effective in ulcerative colitis than in Crohn's disease.

We conducted a six week double blind randomised study of 176 patients with prepyloric gastric ulcer to determine whether the proton pump inhibitor, omeprazole 30 mg daily would accelerate healing and pain relief, as compared with cimetidine 1 g daily. At two, four, and six weeks after entry ulcers healed in a larger percentage of patients treated with omeprazole (54, 81, and 86%) than of those treated with cimetidine (39, 73, and 78%) ('intention to treat' cohort; p less than 0.05 at two weeks). A higher proportion of patients on omeprazole became free of pain during the first week of treatment (p less than 0.05). No major clinical or biochemical side effects were noted. Omeprazole is an efficient treatment for patients with prepyloric gastric ulcers.

One hundred and sixty two patients with endoscopically proved reflux oesophagitis stratified for severity, 66 with grade 1 disease (erythema and friability) and 96 with grade 2 or 3 disease (including erosions or ulcerations), were allocated at random to double blind treatment with omeprazole 40 mg in the morning or ranitidine 150 mg twice daily for up to 12 weeks. A patient could be evaluated sooner if symptomatic relief and endoscopically normal mucosa (grade 0) were noted after four to eight weeks' treatment. Patients treated with omeprazole responded significantly more rapidly than those treated with ranitidine (p less than 0.0001), cumulative healing rates at four, eight, and 12 weeks being 90%, 100%, and 100% respectively for those with grade 1 disease and 70%, 85%, and 91% respectively for those with grade 2 or 3 disease in the omeprazole group. Corresponding rates in the ranitidine group were 55%, 79%, and 88% (grade 1) and 26%, 44%, and 54% (grade 2 or 3). Relief of the major symptoms of heartburn, regurgitation, and dysphagia and improvements in the histological appearance of the mucosa occurred earlier and were again more pronounced during treatment with omeprazole than with ranitidine. This observed superiority of omeprazole 40 mg in the morning over ranitidine 150 mg twice daily in the short term treatment of reflux oesophagitis was obtained without major clinical or biochemical side effects, but further research is needed into longer term use of omeprazole and the effects of the acid inhibition it induces.

Rioprostil, a synthetic 16-methylprostaglandin E1, combines antisecretory with cytoprotective properties, the latter being active even at doses below those required for acid inhibition. To test whether rioprostil given in antisecretory doses would heal prepyloric ulcers rapidly, we assigned patients with endoscopically proved ulcers randomly to double-blind treatment with 100 micrograms rioprostil twice daily or 150 mg ranitidine twice daily for up to 8 weeks. Recruitment was terminated at the time point of planned interim analysis because the total healing rate was markedly lower than expected. Thirty patients were allocated to each treatment group. The cumulative healing rates at 4 and 8 weeks were 40% and 60%, respectively, in the rioprostil group versus 70% and 90%, respectively, in the ranitidine group (p less than 0.01). Pain relief occurred simultaneously in the two groups. No major adverse effects were noted. These findings question the clinical relevance of using 'cytoprotection' by prostaglandin analogues as treatment for prepyloric ulcer disease in the short term.