Mimetic Agonist Research Articles

A WNT mimetic with broad spectrum FZD-specificity decreases fibrosis and improves function in a pulmonary damage model

BackgroundWnt/β-catenin signaling is critical for lung development and AT2 stem cell maintenance in adults, but excessive pathway activation has been associated with pulmonary fibrosis, both in animal models and human diseases such as idiopathic pulmonary fibrosis (IPF). IPF is a detrimental interstitial lung disease, and although two approved drugs limit functional decline, transplantation is the only treatment that extends survival, highlighting the need for regenerative therapies.MethodsUsing our antibody-based platform of Wnt/β-catenin modulators, we investigated the ability of a pathway antagonist and pathway activators to reduce pulmonary fibrosis in the acute bleomycin model, and we tested the ability of a WNT mimetic to affect alveolar organoid cultures.ResultsA WNT mimetic agonist with broad FZD-binding specificity (FZD1,2,5,7,8) potently expanded alveolar organoids. Upon therapeutic dosing, a broad FZD-binding specific Wnt mimetic decreased pulmonary inflammation and fibrosis and increased lung function in the bleomycin model, and it impacted multiple lung cell types in vivo.ConclusionsOur results highlight the unexpected capacity of a WNT mimetic to effect tissue repair after lung damage and support the continued development of Wnt/β-catenin pathway modulation for the treatment of pulmonary fibrosis.

Sialic Acid Mimetic Microglial Sialic Acid-Binding Immunoglobulin-like Lectin Agonism: Potential to Restore Retinal Homeostasis and Regain Visual Function in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD), a leading cause of visual loss and dysfunction worldwide, is a disease initiated by genetic polymorphisms that impair the negative regulation of complement. Proteomic investigation points to altered glycosylation and loss of Siglec-mediated glyco-immune checkpoint parainflammatory and inflammatory homeostasis as the main determinant for the vision impairing complications of macular degeneration. The effect of altered glycosylation on microglial maintained retinal para-inflammatory homeostasis and eventual recruitment and polarization of peripheral blood monocyte-derived macrophages (PBMDMs) into the retina can explain the phenotypic variability seen in this clinically heterogenous disease. Restoring glyco-immune checkpoint control with a sialic acid mimetic agonist targeting microglial/macrophage Siglecs to regain retinal para-inflammatory and inflammatory homeostasis is a promising therapeutic that could halt the progression of and improve visual function in all stages of macular degeneration.

The cold receptor TRPM8 activation in cerebral circulation leads to acute reversible impairment of endothelium‐dependent vasodilator function during selective head cooling

Selective head cooling is frequently used for treatment of hypoxic‐ischemic encephalopathy in newborns. However, the effects of head cooling on cerebral microcirculation are poorly understood. Newborn pigs provide a relevant large animal model of neonatal cerebrovascular regulation. Intravital microscopy using the closed cranial window technique in anesthetized, ventilated newborn pigs of both sexes was implemented to investigate acute effects of selective head cooling on cerebral vascular function. Cerebral vascular function was tested by responses of pial arterioles, the major cerebral resistance vessels, to endothelium‐dependent vasodilator stimuli (systemic hypercapnia and topical bradykinin, glutamate, and hemin), and to sodium nitroprusside (SNP), a nitric oxide donor that produces dilation by targeting arteriolar smooth muscle via a cGMP‐dependent mechanism. Selective head cooling by ice packs lowered the brain and the core temperatures to 25.6 ± 0.3 °C and 33.5 ± 0.1 °C, respectively. Cerebral vascular function was detected consecutively during normothermia, followed by head cooling and rewarming (duration of each period ~ 2h, N=6 pigs). During the hypothermic state, the responses of pial arterioles (mean diameter, 71±5 μm) to endothelium‐dependent vasodilators were greatly reduced, whereas the arteriolar smooth muscle responses to SNP remained intact. After rewarming, all cerebral vascular responses returned to the control normothermia level. We tested the hypothesis that the cold sensing transient receptor potential melastatin channel 8 (TRPM8) which is activated by moderate cold (22–27°C) contributes to impairment of endothelium‐dependent vascular function caused by head cooling. TRPM8 expression in the cerebral endothelium was detected by Western immunoblotting and immunofluorescence techniques. The TRPM8 antagonist AMTB (50 μM) topically applied under the cranial window during the head cooling period completely prevented reduction of endothelium‐dependent cerebral vascular responses (N=5 pigs). The inhibitory effects of head cooling on cerebral vascular function were mimicked by the TRPM8 super‐agonist icilin. Icilin (2 μM) topically applied to cerebral surface during normothermia (core temperature, 37.3 ± 0.2°C) greatly inhibited endothelium‐dependent cerebral vasodilation to hypercapnia, bradykinin, glutamate, and hemin, whereas the dilator responses to SNP were not altered. The ability of icilin to block the endothelium‐dependent dilator responses of pial arterioles during the normothermia period was completely abolished in the presence of the TRPM8 antagonist AMTB (50 μM). Overall, our data demonstrate that hypothermia caused by head cooling acutely and reversibly reduces endothelium‐dependent cerebral vasodilator functions, whereas arteriolar smooth muscle functions remain intact. We provide the first evidence that TRPM8 expressed in the cerebral vascular endothelium is activated by hypothermia or by the cooling mimetic agonists thus leading to acute impairment of endothelium‐dependent regulation of cerebral blood flow during selective head cooling.Support or Funding InformationGrants R01NS101717, R01NS105655 (HP)

Trimebutine, a small molecule mimetic agonist of adhesion molecule L1, contributes to functional recovery after spinal cord injury in mice.

ABSTRACTCuring spinal cord injury (SCI) in mammals is a daunting task because of the lack of permissive mechanisms and strong inhibitory responses at and around the lesion. The neural cell adhesion molecule L1CAM (L1) has been shown to favor axonal regrowth and enhance neuronal survival and synaptic plasticity but delivery of full-length L1 or its extracellular domain could encounter difficulties in translation to therapy in humans. We have, therefore, identified several small organic compounds that bind to L1 and stimulate neuronal survival, neuronal migration and neurite outgrowth in an L1-dependent manner. Here, we assessed the functions of two L1 mimetics, trimebutine and honokiol, in regeneration following SCI in young adult mice. Using the Basso Mouse Scale (BMS) score, we found that ground locomotion in trimebutine-treated mice recovered better than honokiol-treated or vehicle-receiving mice. Enhanced hindlimb locomotor functions in the trimebutine group were observed at 6 weeks after SCI. Immunohistology of the spinal cords rostral and caudal to the lesion site showed reduced areas and intensities of glial fibrillary acidic protein immunoreactivity in both trimebutine and honokiol groups, whereas increased regrowth of axons was observed only in the trimebutine-treated group. Both L1- and L1 mimetic-mediated intracellular signaling cascades in the spinal cord lesion sites were activated by trimebutine and honokiol, with trimebutine being more effective than honokiol. These observations suggest that trimebutine and, to a lesser extent under the present experimental conditions, honokiol have a potential for therapy in regeneration of mammalian spinal cord injuries.

Thrombospondin-1 Mimetic Agonist Peptides Induce Selective Death in Tumor Cells: Design, Synthesis, and Structure-Activity Relationship Studies.

Thrombospondin-1 (TSP-1) is a glycoprotein considered as a key actor within the tumor microenvironment. Its binding to CD47, a cell surface receptor, triggers programmed cell death. Previous studies allowed the identification of 4N1K decapeptide derived from the TSP-1/CD47 binding epitope. Here, we demonstrate that this peptide is able to induce selective apoptosis of various cancer cell lines while sparing normal cells. A structure-activity relationship study led to the design of the first serum stable TSP-1 mimetic agonist peptide able to trigger selective programmed cell death (PCD) of at least lung, breast, and colorectal cancer cells. Altogether, these results will be of valuable interest for further investigation in the design of potent CD47 agonist peptides, opening new perspectives for the development of original anticancer therapies.

Engineering of a Novel Simplified Human Insulin-Like Peptide 5 Agonist.

Insulin-like peptide 5 (INSL5) has recently been discovered as only the second orexigenic gut hormone after ghrelin. As we have previously reported, INSL5 is extremely difficult to assemble and oxidize into its two-chain three-disulfide structure. The focus of this study was to generate structure-activity relationships (SARs) of INSL5 and use it to develop a potent and simpler INSL5 mimetic with RXFP4 agonist activity. A series of human and mouse INSL5 (hINSL5/mINSL5) analogues were designed and chemically synthesized, resulting in a chimeric INSL5 analogue exhibiting more than 10-fold higher potency (0.35 nM) at human RXFP4 compared with native hINSL5 (4.57 nM). The SAR study also identified a key residue (K(A15)) in the A-chain of mINSL5 that contributes to improved RXFP4 affinity and potency of mINSL5 compared with hINSL5. This knowledge ultimately led us to engineer a minimized hINSL5 mimetic agonist that retains native hINSL5-like RXFP4 affinity and potency at human RXFP4. This minimized analogue was synthesized in 17.5-fold higher yield and in less time compared with hINSL5.

Targeting Ischaemia-reperfusion (I-R) Injury in the Intact Heart with Annexin-A1 (ANX-A1) Mimetics

We have recently demonstrated that deficiency of anti-inflammatory protein ANX-A1 exaggerates I-R-induced impairments in myocardial function in the isolated heart. This is the first evidence that ANX-A1 plays an important role in normal cardiac physiology. In this study, we tested the hypothesis that the ANX-A1 mimetics Ac2-26 and Agonist 43 protect myocardial viability and function in the intact heart. Adult male Sprague Dawley rats were Langendorff-perfused at constant flow (10 mL/min) and subjected to 30-min ischaemia/30-min reperfusion ± Ac2-26 (0.3 μM), Agonist 43 (1 μM), or formyl peptide receptor (FPR) one antagonist cyclosporine H (CsH, 1 μM). Adult C57BL/6 mice were subjected to 40-min left arterial descending (LAD) coronary artery ligation/24-h reperfusion, ±ANX-A1 mimetics Ac2-26 (diluent: PBS) or Agonist 43 (diluent: 0.8% Tween in PBS) 5-min before reperfusion. As shown in the table, Ac2-26 reduced myocardial viability and rescues LV function. Although Agonist 43 tended to improve viability, no improvement in LVDP was evident. However, we cannot rule out a contribution of vehicle effect for Agonist 43 in vivo. Our data suggested the non-selective FPR receptor agonist, Ac2-26, maybe more effective in reducing I-R injury in the intact heart, than the small-molecule ANX-A1 mimetic Agonist 43.Tabled 1I-R Ex Vivo (rat)Recovery of LVDP (AUC)LDH Release (AUC)Sham (n = 5)707 ± 46*p<0.05 vs I-R.3 ± 3*p<0.05 vs I-R.I-R (n = 7)320 ± 63494 ± 70I-R + Ac2-26 (0.3 μM, n = 5)636 ± 80*p<0.05 vs I-R.200 ± 55*p<0.05 vs I-R.I-R + Agonist 43 (1 μM, n = 6)414 ± 173184 ± 51*p<0.05 vs I-R.I-R + CsH (1 μM)280 ± 24337 ± 86I-R in vivo (mouse)Infarct size (%)Cardiac troponin (ng/mL)Sham (n = 2)NA0.17I-R (n = 4, i.v.)29 ± 45.74 ± 0.45I-R + Ac2-26 (i.v.) 1 mg/kg, n = 12)18 ± 2*p<0.05 vs I-R.4.54 ± 0.31*p<0.05 vs I-R.I-R + Agonist 43 (i.p., 30 mg/kg, n = 10)19 ± 23.50 ± 0.29*p<0.05 vs I-R.I-R + Vehicle (i.p., 0.8% Tween in PBS)25 ± 53.63 ± 0.08*p<0.05 vs I-R.* p < 0.05 vs I-R. Open table in a new tab

Developing potent backbone cyclic peptides bearing the shared epitope sequence as rheumatoid arthritis drug-leads

Rheumatoid arthritis (RA) is a common human leukocyte antigen-associated disease. Most RA patients have a five-residue sequence motif called the shared epitope (SE) in the DRβ-chain of the HLA-DRB1 protein. The SE was found to activate nitric oxide (NO) production, suggesting a possible mechanism for RA development. The native conformation of the SE is presumed to be an α-helix, thus using cyclic peptides to stabilize this conformation may produce a potent SE mimetic which will have drug-like properties. We present the development of a backbone cyclic SE mimetic that activates NO production in the low nM range. Circular dichroism analysis revealed a conformational change from for the parent linear peptides to the cyclic analogs. The most active cyclic analog is completely stable towards trypsin/chymotrypsin degradation while the linear 15-mer analogs completely degraded within 30 min. The outcome of this study is a potent cyclic peptide with drug-like properties that can be used as a template for drug development.

Neuropeptides of the cotton fleahopper, Pseudatomoscelis seriatus (Reuter)

The cotton fleahopper, Pseudatomoscelis seriatus (Reuter), is an economically important pest of cotton, and increasing concerns over resistance, detrimental effects on beneficial insects and safety issues associated with traditional insecticide applications have led to an interest in research on novel, alternative strategies for control. One such approach requires a more basic understanding of the neurohormonal system that regulates important physiological properties of the fleahopper; e.g. the expression of specific messenger molecules such as neuropeptides. Therefore we performed a peptidomic study of neural tissues from the fleahopper which led to the first identification of the sequences of native peptide hormones. These peptide hormones include the following neuropeptides: corazonin, short neuropeptide F (sNPF), myosuppressin, CAPA-pyrokinin and CAPA-PVK peptides. The CAPA-pyrokinin, sNPF, and CAPA-PVK peptides represent novel sequences. A comparison of fleahopper neuropeptides with those of related heteropteran species indicates that they are quite different. The sNPF of P. seriatus shows, among others, a novel substitution of Leu with Phe within the C-terminal region; a modification that sets it apart from the known sNPFs of not only other Heteroptera but of other arthropod species as well. The identity of the neuropeptides native to the fleahopper can aid in the potential development of biostable, bioavailable mimetic agonists and antagonists capable of disrupting the physiological functions that these neuropeptides regulate.

TWO CALCITONIN-LIKE RECEPTORS EXIST IN THE CHAGAS’ DISEASE VECTOR, RHODNIUS PROLIXUS

Event Abstract Back to Event TWO CALCITONIN-LIKE RECEPTORS EXIST IN THE CHAGAS’ DISEASE VECTOR, RHODNIUS PROLIXUS Meet Zandawala1* and Ian Orchard1 1 University of Toronto Mississauga, Biology, Canada Rhodnius prolixus undergoes a period of rapid diuresis after ingesting large blood meals. Neurohormones with either diuretic or anti-diuretic activity control the process of diuresis by acting on several tissues including the anterior midgut, hindgut and Malpighian tubules. They do so by binding to their receptors. Identification and functional analysis of diuretic factors’ receptors is important as they serve as potential targets for mimetic agonists or antagonists that could be used to disrupt diuresis and ultimately prevent the spread of Chagas’ disease. One of the neurohormones that potentially plays a role in diuresis is Rhopr-diuretic hormone 31 (Rhopr-DH31) which belongs to the insect calcitonin-like family of diuretic hormones. Interestingly, two Rhopr-DH31 receptor-encoding genes exist in R. prolixus (Rhopr-DH31-R1 and Rhopr-DH31-R2), as has been described for several other insect species. Here we report the cDNA sequences of these two receptors and characterize their expression in fifth-instar R. prolixus. Preliminary RT-PCR results demonstrate that Rhopr-DH31-R1 is expressed in multiple tissues, but not the hindgut, in unfed fifth-instar R. prolixus. Highest expression for Rhopr-DH31-R1 is observed in testes, suggesting a potential role for Rhopr-DH31 in male reproduction. However, Rhopr-DH31-R2 is only expressed at very low levels in the central nervous system. Two DH31 receptors have been identified in several insect species and phylogenetic analysis reveals that these receptors could be a result of a gene duplication event since they form two distinct clusters. Acknowledgements Supported by NSERC discovery grant to IO Keywords: Calcitonin-like, DH31, GPCR, insect Conference: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology, Ann Arbor, United States, 13 Jul - 16 Jul, 2011. Presentation Type: Poster Topic: Membrane receptors Citation: Zandawala M and Orchard I (2011). TWO CALCITONIN-LIKE RECEPTORS EXIST IN THE CHAGAS’ DISEASE VECTOR, RHODNIUS PROLIXUS. Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology. doi: 10.3389/conf.fendo.2011.04.00054 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 06 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Mr. Meet Zandawala, University of Toronto Mississauga, Biology, Mississauga, Canada, meet.zandawala@uni-wuerzburg.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Meet Zandawala Ian Orchard Google Meet Zandawala Ian Orchard Google Scholar Meet Zandawala Ian Orchard PubMed Meet Zandawala Ian Orchard Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The nuclear receptor PPARγ individually responds to serotonin- and fatty acid-metabolites

The nuclear receptor, peroxisome proliferator-activated receptor γ (PPARγ), recognizes various synthetic and endogenous ligands by the ligand-binding domain. Fatty-acid metabolites reportedly activate PPARγ through conformational changes of the Ω loop. Here, we report that serotonin metabolites act as endogenous agonists for PPARγ to regulate macrophage function and adipogenesis by directly binding to helix H12. A cyclooxygenase inhibitor, indomethacin, is a mimetic agonist of these metabolites. Crystallographic analyses revealed that an indole acetate functions as a common moiety for the recognition by the sub-pocket near helix H12. Intriguingly, a serotonin metabolite and a fatty-acid metabolite each bind to distinct sub-pockets, and the PPARγ antagonist, T0070907, blocked the fatty-acid agonism, but not that of the serotonin metabolites. Mutational analyses on receptor-mediated transcription and coactivator binding revealed that each metabolite individually uses coregulator and/or heterodimer interfaces in a ligand-type-specific manner. Furthermore, the inhibition of the serotonin metabolism reduced the expression of the endogenous PPARγ-target gene. Collectively, these results suggest a novel agonism, in which PPARγ functions as a multiple sensor in response to distinct metabolites.

Stimulation of N-cadherin-dependent neurite outgrowth by small molecule peptide mimetic agonists of the N-cadherin HAV motif

N-cadherin is a cell adhesion molecule that promotes axon outgrowth and synapse formation during the development of the central nervous system. In addition, N-cadherin promotes glial cell adhesion and myelination of axons. Therefore, stimulating N-cadherin function with N-cadherin agonists could be used therapeutically to promote regeneration of the nervous system and remyelination after injury or disease. In the extracellular domain of N-cadherin, the amino acid sequence HAV is required for N-cadherin-mediated adhesion and neurite outgrowth. The ADH-1 cyclic peptide, derived from the N-cadherin HAV site, is an effective antagonist of N-cadherin-mediated neurite outgrowth and is currently being tested in clinical trials for cancer chemotherapy. Of interest, a dimeric version of this cyclic peptide, N-Ac- CHAVDINGHAVDIC-NH 2, functions as an N-cadherin agonist. This dimeric peptide agonist and the peptide antagonist ADH-1 both have limitations as drugs due to their metabolic instability and lack of oral delivery. To address this issue Adherex Technologies Inc. generated a small molecule library of peptidomimetics to the HAV region of N-cadherin, which would be more amenable to therapeutic use. We screened the Adherex library for compounds that altered neurite outgrowth and identified eight N-cadherin agonists that stimulated N-cadherin-dependent neurite outgrowth. Five of these agonists also stimulated retinal cell migration. These small molecule agonists may be effective reagents for promoting axon growth and remyelination after injury or disease.

Novel 2',6'-dimethyl-L-tyrosine-containing pyrazinone opioid mimetic mu-agonists with potent antinociceptive activity in mice.

Novel bioactive opioid mimetic agonists containing 2',6'-dimethyl-l-tyrosine (Dmt) and a pyrazinone ring interact with mu- and delta-opioid receptors. Compound 1 [3-(4' -Dmt-aminobutyl)-6-(3'-Dmt-aminopropyl)-5-methyl-2(1H)pyrazinone] exhibited high mu-opioid receptor affinity and selectivity (K(i)mu = 0.021 nM and K(i)delta/K(i)mu = 1,519, respectively), and agonist activity on guinea pig ileum (IC(50) = 1.7 nM) with weaker delta-bioactivity on mouse vas deferens (IC(50) = 25.8 nM). Other compounds (2-4) had mu-opioid receptor affinities and selectivities 2- to 5-fold and 4- to 7-fold less than 1, respectively. Intracerebroventricular administration of 1 in mice exhibited potent naloxone reversible antinociception (65 to 71 times greater than morphine) in both tail-flick (TF) and hot-plate (HP) tests. Distinct opioid antagonists had differential effects on antinociception: naltrindole (delta-antagonist) partially blocked antinociception in the TF, but it was ineffective in the HP test, whereas beta-funaltrexamine (irreversible antagonist, mu(1)/mu(2)-subtypes) but not naloxonazine (mu(1)-subtype) inhibited TF test antinociception, yet both blocked antinociception in the HP test. Our data indicated that 1 acted through mu- and delta-opioid receptors to produce spinal antinociception, although primarily through the mu(2)-receptor subtype; however, the mu(1)-receptor subtype dominates supraspinally. Subcutaneous and oral administration indicated that 1 crossed gastrointestinal and blood-brain barriers to produce central nervous system-mediated antinociception. Furthermore, daily s.c. dosing of mice with 1 for 1 week developed tolerance in a similar manner to that of morphine in TF and HP tests, implicating that 1 also acts through a similar mechanism analogous to morphine at mu-opioid receptors.

Cis-peptide bond mimetic tetrazole analogs of the insect kinins identify the active conformation

The insect kinin neuropeptides have been implicated in the regulation of water balance, digestive organ contraction, and energy mobilization in a number of insect species. A previous solution conformation study of an active, restricted-conformation cyclic analog, identified two possible turn conformations as the likely active conformation adopted by the insect kinins at the receptor site. These were a cisPro type VI β-turn over C-terminal pentapeptide core residues 1–4 and a transPro type I-like β-turn over core residues 2–5, present in a ratio of 60:40. Synthesis and evaluation of the diuretic activity of insect kinin analogs incorporating a tetrazole moiety, which mimics a cis peptide bond, identifies the active conformation as the former. The discovery of a receptor interaction model can lead to the development of potent agonist and antagonist analogs of the insect kinins. Indeed, in this study a tetrazole analog with D stereochemistry has been shown to demonstrate partial antagonism of the diuretic activity of natural insect kinins, providing a lead for more potent and effective antagonists of this critical neuropeptide family. The future development of mimetic agonists and antagonists of insect kinin neuropeptides will provide important tools to neuroendocrinologists studying the mechanisms by which they operate and to researchers developing new, environmentally friendly pest insect control strategies.

EPIDURAL EPINEPHRINE INFUSION COMBINED WITH BUPIVACAINE MAY INCREASE BLOOD LOSS DURING LABOR AND DELIVERY

S261 INTRODUCTION: Epidural epinephrine in addition to a local anesthetic can decrease the maternal and fetal peak plasma concentrations of the local anesthetics in obstetrics patients. However, it has been reported that epinephrine containing solution administered into the epidural space decreases uterine activity due to the mimetic agonist effect. Therefore, we examined the effects of epidural epinephrine on the uterine and umbilical blood flow resistance with ultrasonography. We also evaluated the plasma bupivacaine concentrations in mother and fetus, and blood loss during labor and delivery. METHODS: Twenty-eight parturients whose pregnancies were without obstetric problems were enrolled in this study. This study protocol was approved by our hospital's investigation committee on ethics and informed consent was obtained from each patient the day before the study. Labor was induced electively by constant oxytocin infusion under lumber epidural anesthesia. Pregnant women were randomly assigned into two groups after the epidural administration of a bolus of 12ml plus 4ml/hr bupivacaine 0.25% with or without epinephrine (1:200,000). If patients requested additional local anesthetic because of labor pain, we added 6-10 ml of 1% lidocaine. Before induction of epidural analgesia and 20, 60 and 120 min after the first epidural bolus injection, resistance indices of the fetal umbilical artery and the uterine artery were measured with a percutaneous ultrasonography. We also measured the maternal and fetal plasma concentrations of bupivacaine at birth using high performance lipid chromatography, and blood loss during labor and delivery. Data were analyzed using Mann-Whitney U-tests (statistical significance, p<0.05). RESULTS: The resistance index of uterine artery 20 min after bolus injection of bupivacaine was significantly different between the two groups (0.53 +/- 0.08 with epinephrine vs. 0.66 +/- 0.11 without epinephrine, mean +/- SD). However, the resistance indices of fetal umbilical and uterine artery 60 and 120 min after bolus injection of bupivacaine were not significantly different between the two groups. There was also no differences in fetal/maternal bupivacaine concentration ratios between the two groups (0.35 +/- 0.22 with epinephrine vs. 0.41 +/- 0.40 without epinephrine, mean +/- SD). However, the blood loss in the group with epinephrine was significantly more than that without epinephrine (338 +/- 185 g vs. 169 +/- 77 g, mean +/- SD). DISCUSSION: A significant finding of this study was that epidural epinephrine administration combined with bupivacaine increased blood loss during labor and delivery. This may be due to vasodilation caused by the mimetic effects low dose epinephrine (20 [micro sign]g/hr). The utero-placental blood flow resistance was not different between the two groups except for the uterine artery resistance 20 min after bolus injection of bupivacaine. Our results suggest that the low dose epidural epinephrine does not significantly deteriorate uterine activity but may increase blood loss during delivery.

The mechanism of arachidonic acid release in collagen-activated human platelets.

The mechanism of arachidonic acid (AA) release in collagen-activated human platelets was studied. An arachidonic acid metabolite, thromboxane B2 (TXB2), was formed in parallel with the formation of phosphatidic acid (PA) without formation of lysophosphatidic acid (lysoPA) or lysophosphatidylinositol (lysoPI) in the absence of extracellular Ca2+, suggesting that AA was released from PI via a PI-specific phospholipase C (PI-PLC)/diacylglycerol (DG) lipase/monoacylglycerol (MG) lipase pathway under the cytosolic low Ca2+ concentrations. Moreover, solubilized DG lipase and MG lipase could hydrolyze the substrates at basal cytosolic free Ca2+ concentrations. Subsequently, the relationship of cytosolic free Ca2+ concentrations and formation of AA metabolites was analyzed using Ca2+ ionophore, A23187. Collagen was able to induce a release of small amounts of AA under basal cytosolic Ca2+ conditions. However, a release of large amounts of AA was induced by phospholipase A2 activated by both collagen-receptor occupancy and elevated Ca2+ levels. A TXA2 mimetic agonist, STA2 induced all the responses except for AA release. From these results, the mechanism of AA release and signal transduction in collagen-activated human platelets is discussed.

Acyl, pseudotetra‐, tri‐ and dipeptide active‐core analogs of insect neuropeptides

Pseudopeptides of the achetakinin insect neuropeptide family were synthesized by replacing the amino acid blocks Phe-, Phe-Tyr-, and Phe-Tyr-Pro- of the active-core pentapeptide Phe-Tyr-Pro-Trp-Gly-NH2 with hydrocinnamic acid, 6-phenylhexanoic acid, and both 9-phenylnonanoic and 6-phenylhexanoic acid, respectively. All four of these analogs retained myotropic activity, demonstrating that the active core could be reduced from a pentapeptide to a modified dipeptide. Most notable of these was the pseudotetrapeptide hydrocinnamyl-Tyr-Pro-Trp-Gly-NH2, which retained 70% of the potency and over 85% of the maximal activity of the parent pentapeptide. The N-terminal amino group, the phenol ring of the Tyr residue, the sulfate moiety and the Gly residue of the insect sulfakinin active core Tyr(SO3H)-Gly-His-Met-Arg-Phe-NH2 were all replaced by dodecanedioic acid. The resulting pseudotetrapeptide, dodecandioyl-His-Nle-Arg-Phe-NH2, elicited myostimulatory activity. Conversely, the related acyl pseudopentapeptide azelayl-Gly-His-Nle-Arg-Phe-NH2 proved myoinhibitory. A possible explanation for these disparate biological responses is discussed. These acyl pseudopeptides are important advances towards the eventual development of stable, potent mimetic agonists and antagonists of insect neuropeptides.

Activation of phospholipases in platelets by polyclonal antibodies against a surface membrane protein

In a previous paper we demonstrated using immunochemical techniques that propolypeptide of von Willebrand factor was present on the surface of resting platelets. In the present paper we show that polyclonal antibodies against propolypeptide of von Willebrand factor induce activation of phospholipase(s) in platelets and lead to platelet aggregation. The antibody-stimulation of platelets induced the synthesis of thromboxane A 2 (TXA 2). Furthermore, the aggregation was inhibited by aspirin and an antagonist of TXA 2. Aspirin inhibited not only the aggregation but also the activation of arachidonic acid liberation from phospholipids, but the effect of aspirin on arachidonic acid liberation was cancelled by the combined effect of the antibodies and a TXA 2 mimetic agonist, which itself did not activate arachidonic acid liberation. The antibody-induced activation of arachidonic acid liberation and the aggregation were blocked by cytochalasin B. All these results obtained with antibodies were quite similar to the results obtained with collagen.

Thromboxane A 2 mimetic U46619 stimulates ciliary motility of rabbit tracheal epithelial cells

To elucidate whether thromboxane A 2 (TxA 2), one of the important arachidonic acid metabolites that may play a role ion the development of airway inflammation, affects respiratory ciliary motility and, if so, what the mechanism of action is, we measured ciliary beat frequency (CBF) of rabbit cultured tracheal epithelium in response to U46619, a TxA 2 mimetic agonist, by a photoelectric method. Addition of U46619 (10 −5 M) increased CBF from 17.7 ± 0.7 to 22.8 ± 1.4 Hz ( mean ± SE, p < 0.01) within 5 min, which was followed by a decline to the baseline value by 10 min. This effect was concentration-dependent, the maximal increase from the baseline value and the drug concentration required to produce a half-maximal effect (EC 50) being 26.9 ± 4.6% ( p < 0.01) and 3 × 10 −7 M, respectively. The U46619-induced increase in CBF was abolished by SQ29548, a TxA 2 receptor antagonist, and inhibited by verapamil, a Ca 2+-entry blocker, and H-7, a protein kinase C inhibitor. These results suggest that TxA 2 stimulates ciliary motility through the activation of airway epithelial TxA 2 receptors, and that this effect may be exerted from Ca 2+-influx and protein kinase C.

Toward nonpeptidal substance P mimetic analogues: Design, synthesis, and biological activity

1,4-Piperazine and 4-hydroxyproline, two small cyclic polyfunctional systems with defined stereochemistry, were introduced as "molecular scaffolds." We define a "bioactive topology," which is a derived putative low-energy conformation obtained through theoretical conformational analysis of substance P. Substitution of these molecular scaffolds by pharmacophors characteristic of the bioactive topology of the C-terminal hexapeptide of substance P resulted in active, partially nonpeptidal substance P mimetic agonists. The study discusses the concepts and tools used to achieve this structural transformation, and points out the need to address flexibility-rigidity issues in an attempt to maintain sufficient molecular plasticity.