scholarly journals Evaluation of [Cys(ATTO 488)8]Dermorphin-NH2 as a novel tool for the study of μ-opioid peptide receptors

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250011
Author(s):  
Despina Giakomidi ◽  
Mark F. Bird ◽  
John McDonald ◽  
Erika Marzola ◽  
Remo Guerrini ◽  
...  
Keyword(s):  
Functional Activity ◽  
Radioligand Binding ◽  
Opioid Peptide ◽  
Confocal Scanning Laser Microscopy ◽  
Dependent Manner ◽  
Orphanin Fq ◽  
Concentration Dependent Manner ◽  
Whole Cells ◽  
Hek Cells ◽  
Confocal Scanning Laser

The μ-opioid peptide (MOP) receptor is a member of the opioid receptor family and an important clinical target for analgesia. Measuring MOP receptor location and tracking its turnover traditionally used radiolabels or antibodies with attendant problems of utility of radiolabels in whole cells and poor antibody selectivity. To address these issues we have synthesized and characterised a novel ATTO488 based fluorescent Dermorphin analogue; [Cys(ATTO 488)8]Dermorphin-NH2 (DermATTO488). We initially assessed the binding profile of DermATTO488 in HEK cells expressing human MOP and CHO cells expressing human MOP, δ-opioid peptide (DOP), κ-opioid peptide (KOP) and Nociceptin/Orphanin FQ peptide (NOP) receptors using radioligand binding. Functional activity of the conjugated peptide was assessed by measuring (i) the ability of the ligand to engage G-protein by measuring the ability to stimulate GTPγ[35S] binding and (ii) the ability to stimulate phosphorylation of ERK1/2. Receptor location was visualised using confocal scanning laser microscopy. Dermorphin and DermATTO488 bound to HEKMOP (pKi: 8.29 and 7.00; p<0.05), CHOMOP (pKi: 9.26 and 8.12; p<0.05) and CHODOP (pKi: 7.03 and 7.16; p>0.05). Both ligands were inactive at KOP and NOP. Dermorphin and DermATTO488 stimulated the binding of GTPγ[35S] with similar pEC50 (7.84 and 7.62; p>0.05) and Emax (1.52 and 1.34fold p>0.05) values. Moreover, Dermorphin and DermATTO488 produced a monophasic stimulation of ERK1/2 phosphorylation peaking at 5mins (6.98 and 7.64-fold; p>0.05). Finally, in confocal microscopy DermATTO488 bound to recombinant MOP receptors on CHO and HEK cells in a concentration dependent manner that could be blocked by pre-incubation with unlabelled Dermorphin or Naloxone. Collectively, addition to ATTO488 to Dermorphin produced a ligand not dissimilar to Dermorphin; with ~10fold selectivity over DOP. This new ligand DermATTO488 retained functional activity and could be used to visualise MOP receptor location.

scholarly journals Effects of Sizes and Conformations of Fish-Scale Collagen Peptides on Facial Skin Qualities and Transdermal Penetration Efficiency

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Huey-Jine Chai ◽  
Jing-Hua Li ◽  
Han-Ning Huang ◽  
Tsung-Lin Li ◽  
Yi-Lin Chan ◽  
...  
Keyword(s):  
Cell Model ◽  
Confocal Scanning Laser Microscopy ◽  
Dependent Manner ◽  
Fish Scale ◽  
Collagen Peptides ◽  
Confocal Scanning ◽  
Transdermal Penetration ◽  
Confocal Scanning Laser ◽  
The Given ◽  
Dose Dependent

Fish-scale collagen peptides (FSCPs) were prepared using a given combination of proteases to hydrolyze tilapia (Oreochromissp.) scales. FSCPs were determined to stimulate fibroblast cells proliferation and procollagen synthesis in a time- and dose-dependent manner. The transdermal penetration capabilities of the fractionationed FSCPs were evaluated using the Franz-type diffusion cell model. The heavier FSCPs, 3500 and 4500 Da, showed higher cumulative penetration capability as opposed to the lighter FSCPs, 2000 and 1300 Da. In addition, the heavier seemed to preserve favorable coiled structures comparing to the lighter that presents mainly as linear under confocal scanning laser microscopy. FSCPs, particularly the heavier, were concluded to efficiently penetrate stratum corneum to epidermis and dermis, activate fibroblasts, and accelerate collagen synthesis. The heavier outweighs the lighter in transdermal penetration likely as a result of preserving the given desired structure feature.

scholarly journals Thromboxane A2 receptor is highly expressed in mouse immature thymocytes and mediates DNA fragmentation and apoptosis.

1993 ◽  
Vol 178 (5) ◽  
pp. 1825-1830 ◽  
Author(s):  
F Ushikubi ◽  
Y Aiba ◽  
K Nakamura ◽  
T Namba ◽  
M Hirata ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Radioligand Binding ◽  
Apoptotic Cell ◽  
Blood Platelets ◽  
Dependent Manner ◽  
Receptor Density ◽  
Concentration Dependent Manner ◽  
Receptor System ◽  
Txa2 Receptor ◽  
Immature Thymocytes

We have recently revealed that the thymus is the organ showing the highest expression of thromboxane (TX) A2 receptor in mice. In this study, thymic cell populations expressing the receptor were identified, and the effects of a TXA2 agonist on these cells were examined. Radioligand binding using a TXA2 receptor-specific radioligand revealed a single class of binding sites in the thymocytes with an affinity and specificity identical to those reported for the TXA2 receptor. The receptor density in these cells was comparable to that seen in blood platelets. This receptor is most highly expressed in CD4-8- and CD4+8+ immature thymocytes, followed by CD4+8- and CD4-8+ cells. The receptor density in splenic T cells was less than one fifth of that in CD4+8+ cells and no binding activity was detectable in splenic B cells. The addition of a TXA2 agonist, STA2, to thymocytes induced the disappearance of the CD4+8+ cells in a time- and concentration-dependent manner and caused DNA fragmentation. These changes were blocked by a specific TXA2 antagonist, S-145. These results demonstrate that TXA2 induces apoptotic cell death in immature thymocytes by acting on the TXA2 receptor on their cell surface and suggest a role for the TXA2/TXA2 receptor system in the thymic micro-environment.

The Effect Of Thrombin On The Electrophoretic Mobility And Functional Activity Of Various Factor VIII Molecular Forms

1981 ◽  
Author(s):  
M J Seghatchian ◽  
I I Mackie
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Conformational Changes ◽  
Functional Activity ◽  
Gel Electrophoresis ◽  
Molecular Forms ◽  
Agarose Gel ◽  
Dependent Manner ◽  
Agarose Gel Electrophoresis ◽  
Concentration Dependent Manner

Several distinct populations of factor VIII procoagulant activity (VIIIC) were observed in some clinical concentrates after agarose gel electrophoresis. It is not yet known whether these different forms are the products of proteolytic digestion, or subtle conformational changes of FVIII structure, which may occur during concentrate preparation. Low thrombin concentrations activate FVIII in a time/concentration dependent manner, and the 2 proteins have high affinities for one another. We have studied the properties of the various FVIII forms before and after incubation of concentrate with highly purified α-thrombin. Agarose gel electrophoresis was used to monitor: a) quantitative changes in functional activity (by 2 stage clotting (VIII:C) and amidolytic (VIII: CAm) assays); b) the binding of 125I- anti VIII:C (*IgG) to FVIII: C; c) the crossed immunoelec- trophoretic (CIE) pattern of FVIII: RAg.In the absence of thrombin, FVIII concentrate showed 2 main populations of FVIII: C and VIII: CAm. The radioactivity (VIII: CAg) pattern correlated with the slower moving of these populations. Low thrombin concentrations (0.0001-0.01 u/nl) caused no detectable change in FVIII: C level in the slow peak but additional peaks of activity appeared at the sample well, and between the well and slow peak. This was accompanied by a decrease in the fast moving peak, and the appearance of a third new peak with slightly less mobility. Higher thrombin concentrations (0.5u/ml) caused a marked decrease in the slow moving peak, with a concomitant increase in the well peak, by all methods. Increasing thrombin concentration progressively reduced *IgG binding, and a pattern similar to serum was obtained. Thrombin caused a slightly faster migration of FVIII: RAg, a well peak was sometimes seen. Thrombin therefore appeared to act preferentially on the lower molecular weight FVIII activity, and the high molecular weight peaks that occurred may represent complexes of FVIII with thrombin and/or other contaminant proteins present in concentrate such as fibrinogen or fibronectin.

Physiological effects of FMRFamide-related peptides and classical transmitters on dispersed muscle fibres of the turbellarian, Procerodes littoralis

Parasitology ◽  
2001 ◽  
Vol 122 (4) ◽  
pp. 447-455 ◽  
Author(s):  
C. G. MONEYPENNY ◽  
N. KRESHCHENKO ◽  
C. L. MOFFETT ◽  
D. W. HALTON ◽  
T. A. DAY ◽  
...  
Keyword(s):  
Muscle Contraction ◽  
Confocal Scanning Laser Microscopy ◽  
Maximal Response ◽  
Muscle Fibres ◽  
Dependent Manner ◽  
Physiological Effects ◽  
Concentration Dependent Manner ◽  
Muscle System ◽  
High K ◽  
Procerodes Littoralis

The physiological effects of selected classical transmitters and FMRFamide-related peptides (FaRPs) on dispersed muscle fibres from the marine turbellarian, Procerodes littoralis have been examined. Confocal scanning laser microscopy coupled with fluorescein isothiocyanate (FITC) or tetramethylrhodamine (TRITC)-labelled phalloidin revealed a highly developed body wall muscle system with circular, longitudinal and diagonal layers of muscle fibres. Dispersed muscle fibres contracted when depolarized by exposure to extracellular media with elevated K+ (15–100 mM) in a concentration-dependent manner, with a maximal response of 87% achieved at [ges ] 75 mM. 5-Hydroxytryptamine (5-HT) induced concentration-dependent muscle contraction between 0·01 and 1000 μM, with 10 μM producing a near maximal contraction response of 75%. Acetylcholine (ACh) had less pronounced excitatory effects (0·01–1000 μM), inducing contraction of only 32% of the fibres at 100 μM. The flatworm FMRFamide-related peptides (FaRPs), GYIRFamide, YIRFamide and GNFFRFamide each had concentration-dependent myocontractile effects, indicating the occurrence of at least 1 FaRP receptor on P. littoralis muscle fibres. At 10 μM peptide, GNFFRFamide induced contractions in < 40% of the muscle fibres examined, whereas YIRFamide and GYIRFamide induced contraction in 70 and 75%of muscle fibres, respectively. The order of potency of the peptides was: GYIRFamide > YIRFamide > GNFFRFamide. Pre-incubation of the muscle fibres in 5 μM 5-HT significantly reduced the responses to GYIRFamide, YIRFamide and 5-HT, while the responses to high K+ remained unaltered. Muscle fibres pre-incubated in GYIRFamide (0·1 μM) were also less responsive to 5-HT but not to ACh and high-K+. The GYIRFamide analogue, GYIRdFamide, did not induce muscle contraction (0·01–100 μM) per se, but when co-applied with the myoactive peptides GYIRFamide, YIRFamide or GNFFRFamide, it significantly blocked their ability to elicit contractions. This suggests that the peptides tested may act via a common muscle-based neuropeptide receptor. GYIRdFamide did not alter the contractile effects of high K+, 5-HT or ACh. Collectively, these results indicate that FaRPs, 5-HT and ACh all have the potential to cause muscle contraction in flatworms and that 5-HT and FaRPs alter muscle sensitivity to each other, but do not influence the ability of flatworm muscle fibres to contract.

scholarly journals Alloxan inhibits ligand binding to adrenoceptors of vascular smooth muscle microsomes

1990 ◽  
Vol 270 (1) ◽  
pp. 137-140 ◽  
Author(s):  
C Y Kwan ◽  
S Sipos ◽  
V Gaspar
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Vascular Smooth Muscle ◽  
Radioligand Binding ◽  
Mesenteric Arteries ◽  
Dependent Manner ◽  
Inhibitory Effects ◽  
Experimental Conditions ◽  
Concentration Dependent Manner ◽  
Number Of Binding Sites

We have examined the effects of alloxan on the binding of [3H]prazosin and [125I]monoiodocyanopindolol (ICYP) to plasma-membrane-enriched microsomes isolated from dog aortas and dog mesenteric arteries respectively. Preincubation of the vascular smooth muscle membranes with alloxan reduced the number of binding sites of the alpha- and β-adrenoceptors in a concentration-dependent manner, whereas the affinity of the radioligands for the adrenoceptors was not affected by alloxan. Streptozotocin, which is also a diabetogenic agent like alloxan, had no effect on the radioligand binding to these adrenoceptors under similar experimental conditions. The inhibitory effects of alloxan on binding to β-adrenoceptors were found to be highly pH-dependent. These results indicate that alloxan exerts adverse effects on cell membrane adrenoceptors in addition to those on the ion-transport function of vascular smooth muscle cell [Kwan (1988) Biochem. J. 254, 293-296], and also suggest that the primary site of action of alloxan is the plasma membrane.

Interaction of amiloride with α-adrenoreceptors: evidence from radioligand binding studies

1988 ◽  
Vol 66 (5) ◽  
pp. 596-600 ◽  
Author(s):  
S. M. Periyasamy
Keyword(s):  
Radioligand Binding ◽  
Rat Kidney ◽  
Rat Tissues ◽  
Dependent Manner ◽  
Diuretic Effect ◽  
Regulatory Effect ◽  
Binding Studies ◽  
Concentration Dependent Manner ◽  
Mammalian Tissues ◽  
Radioligand Binding Studies

We investigated the effect of amiloride on α-adrenoreceptors (α1 and α2) using radioligand binding techniques. Amiloride inhibited [3H]yohimbine and [3H]prazosin binding to α2- and α1-adrenoreceptors, respectively, from various tissues in a concentration-dependent manner. Amiloride was approximately 9–12 times more potent in inhibiting [3H]yohimbine binding to α2-adrenoreceptors from rat tissues than from other mammalian tissues. However, it had almost the same potency in inhibiting [3H]prazosin binding to α1-adrenoreceptors from rat as well as other mammalian tissues. Further, in rat tissues, amiloride was approximately 10 times more potent in inhibiting [3H]yohimbine than [3H]prazosin binding. Amiloride inhibited [3H]yohimbine binding noncompetitively and [3H]prazosin binding competitively. The inhibition of [3H]yohimbine and [3H]prazosin binding by amiloride was reversible. Since amiloride has been shown to be an inhibitor of Na+–H+ exchanger protein, we believe that it regulates the α2-adrenoreceptors by binding to Na+–H+ exchanger protein. Triamterene, a compound similar to amiloride in regard to diuretic effect, had very little effect on [3H]yohimbine and [3H]prazosin binding to rat kidney membranes, suggesting that the α-adrenoreceptor antagonistic properties of amiloride are not related to its antikaliuretic effect. The results of the present study suggest that some of the pharmacological actions of amiloride (antihypertensive and diuretic effects) can be explained in part by its regulatory effect on both α1- and α2-adrenoreceptors.

Cionin, a protochordean hybrid of cholecystokinin and gastrin: biological activity in mammalian systems

1991 ◽  
Vol 260 (6) ◽  
pp. G977-G982
Author(s):  
Birgit Schjoldager ◽  
Jung Park ◽  
Anders H. Johnsen ◽  
Tadataka Yamada ◽  
Jens F. Rehfeld
Keyword(s):  
Common Ancestor ◽  
Radioligand Binding ◽  
Somatostatin Receptors ◽  
Gallbladder Motility ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cell Content ◽  
Somatostatin Cells ◽  
Equilibrium Data ◽  
Best Fit

The protochordean octapeptide cionin is structurally a hybrid of mammalian cholecystokinin (CCK) and gastrin, and thus their possible common ancestor. To determine whether cionin behaves like CCK or gastrin, we examined its effect on canine fundic somatostatin cells and on porcine and bovine gallbladder muscles. Cionin released somatostatin with a potency (ED50 0.15 nM) and efficacy (14.8% of cell content) similar to that of CCK-8 (ED50 0.12 nM, efficacy 16.7%). The efficacies but not the potencies of CCK-8 and cionin differed from those of sulfated gastrin (0.12 nM, 9.7%), nonsulfated gastrin (0.20 nM, 9.4%), and nonsulfated CCK-8 (0.30 nM, 10.4%). CCK and gastrin stimulated contractions of porcine gallbladder muscle strips in a concentration-dependent manner with no differences in efficacy but with characteristic differences in potency. CCK-8 and cionin displayed similar potencies of ED50 2.0 and 2.6 nM; both were significantly different from the ED50 of 0.4 μM for sulfated gastrin and 2.3 μM for nonsulfated gastrin. CCK radioligand binding to membrane-enriched preparations of porcine and bovine gallbladder muscularis was specific and of high affinity. The equilibrium data revealed that binding of CCK and gastrin peptides best fit a single site. CCK-8 and cionin displayed similar affinities [Kd 0.5 nM (porcine), 0.5 nM (bovine, CCK) vs. Kd 0.8 and 0.9 nM (cionin), respectively]. These differed again significantly from Kd 0.6 and 1.5 μM (sulfated gastrin) and 0.7 and 0.2 μM (nonsulfated gastrin). The results show that cionin behaves like CCK rather than gastrin in mammals. gallbladder motility; somatostatin; receptors Submitted on August 10, 1990 Accepted on December 19, 1990

Computer-Assisted Analysis of Multi-Color Confocal Microscopic Datasets: Automated Light Microscopic Discrimination of Synaptic Relationships

1996 ◽  
Vol 54 ◽  
pp. 284-285
Author(s):  
M Wessendorf ◽  
A Beuning ◽  
D Cameron ◽  
J Williams ◽  
C Knox
Keyword(s):  
Nerve Fibers ◽  
Confocal Scanning Laser Microscopy ◽  
Computer Assisted ◽  
Nerve Terminals ◽  
Neuronal Somata ◽  
Scanning Laser Microscopy ◽  
Confocal Scanning ◽  
Entire Cell ◽  
Confocal Scanning Laser ◽  
Assisted Analysis

Multi-color confocal scanning-laser microscopy (CSLM) allows examination of the relationships between neuronal somata and the nerve fibers surrounding them at sub-micron resolution in x,y, and z. Given these properties, it should be possible to use multi-color CSLM to identify relationships that might be synapses and eliminate those that are clearly too distant to be synapses. In previous studies of this type, pairs of images (e.g., red and green images for tissue stained with rhodamine and fluorescein) have been merged and examined for nerve terminals that appose a stained cell (see, for instance, Mason et al.). The above method suffers from two disadvantages, though. First, although it is possible to recognize appositions in which the varicosity abuts the cell in the x or y axes, it is more difficult to recognize them if the apposition is oriented at all in the z-axis—e.g., if the varicosity lies above or below the neuron rather than next to it. Second, using this method to identify potential appositions over an entire cell is time-consuming and tedious.

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak
Keyword(s):  
Neutrophil Elastase ◽  
Cell Activation ◽  
Thromboxane A2 ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

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