Structural and functional characterization of an egg-laying hormone signaling system in a lophotrochozoan - The pacific oyster (Crassostrea gigas).

The egg-laying hormones (ELHs) of gastropod mollusks were characterized more than forty years ago. Yet, they have remained little explored in other mollusks. To gain insights into the functionality of the ELH signaling system in a bivalve mollusk - the oyster Crassostrea gigas, this study investigates the processing of its ELH precursor (Cragi-ELH) by mass spectrometry. Some of the ELH mature peptides identified in this study were subsequently investigated by nuclear magnetic resonance and shown to adopt an extended alpha-helix structure in a micellar medium mimicking the plasma membrane. To further characterize the ELH signaling system in C. gigas, a G protein-coupled receptor phylogenetically related to ecdysozoan diuretic hormone DH44 and corticotropin-releasing hormone (CRH) receptors named Cragi-ELHR was also characterized functionally and shown to be specifically activated by the two predicted mature ELH peptides and their N-terminal fragments. Both Cragi-ELH and Cragi-ELHR encoding genes were mostly expressed in the visceral ganglia (VG). Cragi-ELH expression was significantly increased in the VG of both fully mature male and female oysters at the spawning stage. When the oysters were submitted to a nutritional or hyposaline stress, no change in the expression of the ligand or receptor genes was recorded, except for Cragi-ELHR only during a mild acclimation episode to brackish water. These results suggest a role of Cragi-ELH signaling in the regulation of reproduction but not in mediating the stress response in our experimental conditions.

Understanding the Effect of Thermal Treatment on the Physico-Mechanical Properties of Light-Cured Composites for use in Indirect Restorations.

OBJECTIVES: To study the potential benefits of a post-cure thermal treatment on key physico-mechanical properties of light-cured resin-based composites for use in indirect restorations, a CAD/CAM composite block being used as control. MATERIAL AND METHODS: Six commercial composites were light-cured before being thermally treated in a furnace at 90°C during 15 minutes (CAD/CAM composite used as a control). The properties measured with or without thermal treatment were: degree of conversion, flexural strength, elastic modulus, Vickers microHardness, organic mass content and eluted and absorbed mass before and after storage in ethanol. The data were analysed using one-way ANOVA, and Weibull distributions. RESULTS: A general increase in the properties measured was observed for all materials after thermal treatment, except a general decrease in mass elution and absorption (most statistically significant: p⟨0.05). Weibull analysis showed a tendency (p⟩0.05) of increased reliability of the flexural strength after thermal treatment for all materials. CONCLUSION: The present data revealed clear physico-mechanical improvements after thermal treatment of light-cured composites. Such method could hence be beneficially used to produce indirect restorations as compared to stratifying and light-curing the same composites in situ. However, most properties of the control CAD/CAM composite were higher, but CAD/CAM technologies aren't available everywhere.

Gonadotropin-inhibitory hormone in teleosts: New insights from a basal representative, the eel.

Since its discovery in birds, gonadotropin-inhibitory hormone (GnIH) has triggered investigation in the other groups of vertebrates. In the present study, we have identified a single gnih gene in the European eel (Anguilla anguilla), a representative species of a basal group of teleosts (Elopomorphs). We have also retrieved a single gnih gene in Osteoglossomorphs, as well as in more recently emerged teleosts, Clupeocephala. Phylogeny and synteny analyses allowed us to infer that one of the two gnih paralogs emerged from the teleost-specific whole genome duplication (TWGD or 3R), would have been lost shortly after the 3R, before the emergence of the basal groups of teleosts. This led to the presence of a single gnih in extant teleosts as in other vertebrates. Two gnih paralogs were still found in some teleost species, such as in salmonids, but resulting from the additional whole genome duplication that specifically occurred in this lineage (4R). Eel gnih was mostly expressed in the diencephalon part of the brain, as analyzed by quantitative real-time PCR. Cloning of eel gnih cDNA confirmed that the sequence of the GnIH precursor encoded three putative mature GnIH peptides (aaGnIH-1, aaGnIH-2 and aaGnIH-3), which were synthesized and tested for their direct effects on eel pituitary cells in vitro. Eel GnIH peptides inhibited the expression of gonadotropin subunits (lhß, fshß, and common a-subunit) as well as of GnRH receptor (gnrh-r2), with no effect on tshß and gh expression. The inhibitory effect of GnIH peptides on gonadotropic function in a basal teleost is in agreement with an ancestral inhibitory role of GnIH in the neuroendocrine control of reproduction in vertebrates.

Exploring the influence of placing bi-directional E-glass fibers as protective layer under a CAD-CAM resin composite on the fracture pattern.

OBJECTIVES: To investigate the influence of the presence and position of bidirectional E-glass fibers under a CAD-CAM resin composite on the fracture pattern evaluated both after quasi-static mechanical loading and after fatigue. METHODS: Rectangular specimens (10 mm-long, 5 mm-large and 4.2 mm-thick) were prepared and divided into four groups (n = 30/group). The control group (C-Group) consisted of a 4.2 mm-thick layer of monolithic CAD/CAM resin composite resin (Cerasmart, GC). In the 3 other groups including the placement of a fiber layer (F-Groups), the CAD/CAM resin composite layer was reduced to 3-, 2- and 1-mm thickness (F3-, F2- and F1-Groups, respectively). Two bonded layers of bidirectional E-glass FRC (Dentapreg, ADM A.S.) were bonded underneath and a light-curable resin composite base (Gaenial Posterior, GC) was then added to reach a total thickness of 4.2 mm for all samples. In each group, half of the specimens (n = 15) were submitted to quasi-static mechanical loading to failure in a universal testing machine. The other half (n = 15) was subjected to cyclic isometric stepwise loading until failure or completion of 105000 cycles (5000 cycles at 500 N, followed by five stages of 20000 cycles at 750 N, 1000 N, 1250 N, 1500 N, and 1750 N). The data were analyzed by Weibull statistics for quasi-static loading, and by the Kaplan-Meier product limit estimation procedure after fatigue. All fractured specimens were studied using light and electron microscopy techniques, and the types of fracture were determined. RESULTS: For quasi-static mechanical loading, significant differences were observed for Weibull modulus and characteristic strength between groups, with values ranging from 10.8 to 22.4 for the former and from 2336.6 to 2974.7 for the latter. Also, survival after stepwise fatigue revealed statistically significant differences between groups (p < 0.05), the lowest values of cycles before failure being observed for F1-Group - Median = 61223 (50415; 65446) - as compared to the other groups - C-Group: Median = 89005 (86189; 98195); F3-Group: Median = 85198 (77279; 87860); F2-Group: Median = 89306 (87454; 97024). Both in quasi-static loading and after fatigue, the observation of fracture modes revealed major differences. While all fractures were vertical (split) in C-Group, the majority of the specimens in F-Groups presented some degree of horizontal deflection of the crack. In all deviated fractures, fractographic analysis confirmed a toughening effect of the fiber layer. SIGNIFICANCE: The present in vitro work tends to show that the fracture pattern of CAD-CAM resin composites is favorably affected by the presence and position of an underlying bidirectional E-glass fiber layer. The placement of E-glass fibers under a CAD-CAM resin composite may therefore represent an interesting strategy to reduce the risk of catastrophic restoration failure, which could be integrated in the development of the new generation of indirect materials, possibly in 3D-printing approaches.

Interactions between immune system and mesenchymal stem cells in dental pulp and periapical tissues.

The recent isolation and characterization of mesenchymal stem cells (MSCs) in dental tissues constitutes a major step forward in the development of new treatment strategies. MSCs are essential for dental pulp repair and the success of regenerative endodontic procedures. It is important to understand that immune cells and cytokines can affect stem cell function, which can impact their healing potential. On the other hand, stem cells are immunoprivileged and have the ability to modulate immune and inflammatory responses, which can be utilized to improve treatments outcome. This review addresses both aspects of this interaction and suggests that any change on both sides can tip the balance in favour of either persistence of inflammation or healing. Finally, the therapeutic relevance of the interaction between MSCs and immune system relative to current treatments is discussed, and future research and treatment perspectives are suggested.

Quality of Cure in Depth of Commercially Available Bulk-fill Composites: A Layer-by-layer Mechanical and Biological Evaluation.

Despite their popularity, the use of bulk-fill composites remains controversial, both in terms of their properties and their in-depth development. The objectives of the present work were (1) to provide a more comprehensive evaluation of the quality of cure in depth of commercially available bulk-fill composites by combining various key mechanical and biological characterization methods, (2) to evaluate the inter-material differences when optimally cured, and (3) to evaluate the efficiency of an antioxidant-N-acetyl-cysteine (NAC)-to restrain the adverse effects of the leached components on cell viability. Nine bulk-fill composites (including flowable and high-viscosity materials) were investigated and compared to two conventional resin-based composites, one flowable and one high-viscosity restorative material. The materials were injected or packed into Teflon molds of various configurations, up to 6 mm material thickness. They were then light-cured from the top for 20 seconds with Bluephase G2 (Ivoclar Vivadent, irradiance = 1050 mW/cm2). The following physico-mechanical properties were measured for the upper (0-2 mm), intermediate (2-4 mm), and lower (4-6 mm) layers: degree of conversion using Raman Spectrometry (DC, in %), microhardness using a Vickers micro-indenter before (VHN dry) and after 24 hours of storage in ethanol (VHN EtOH), and flexural strength (in MPa) and flexural modulus (in GPa) using a three-point bend test. Each composite layer and an uncured layer were also stored for one week in a standard cell growth medium to generate conditioned media. Human dental pulp cells were then cultured for 24 hours with the latter and cell viability was measured using an MTS assay. A similar experiment was repeated with conditioned media produced in contact with uncured composites, with and without the addition of 4 mM NAC. The data were subjected to a Shapiro-Wilk test, then one-way ANOVA or Kruskal-Wallis test, followed either by Tukey's test (inter-material comparison) or by Dunnett's or Dunn's test (comparison between layers relative to the upper one). The level of statistical significance was set at 0.05. Some materials (EverX, X-traF, VenusBF, X-traB) did not show any significant differences (p>0.05) for any of the properties considered between the intermediate layers compared to the upper one (considered as reference). Others displayed significant differences, at least for some properties, highlighting the value of combining various key mechanical and biological characterization methods when investigating the quality of cure in depth. Significant inter-material differences (p<0.05) were observed when comparing the properties of their upper layer, considered as "optimally" polymerized. Hence, one needs to consider the absolute property values, not only their relative evolution concerning layer thickness. Finally, the use of NAC appeared as beneficial to reduce the risk of harmful effects to dental pulp cells, especially in case of excessive thickness use, and may therefore be of potential interest as an additive to composites in the future.

First evidence for a direct inhibitory effect of kisspeptins on LH expression in the eel, Anguilla anguilla.

The kisspeptin system has emerged as one of the main puberty gatekeepers among vertebrates. The European eel (Anguilla anguilla) is a remarkable model due to its phylogenetical position at the basis of teleosts, and its unique life cycle with a blockade of puberty before reproductive migration. We cloned the full-length coding sequence of a kisspeptin receptor (Kissr) in the eel. Comparison of Kissr sequences assigned the eel Kissr to a basal position in a clade including most of the known teleost Kissr, in agreement with the eel phylogenetical position. Eel Kissr tissue distribution was analyzed by quantitative real-time PCR. Eel Kissr was highly expressed in the brain, especially in the telencephalon and di-/mes-encephalon, while a very low or undetectable expression was observed in various peripheral organs. A high expression of Kissr was also found in the pituitary indicating a possible direct pituitary role of kisspeptin. Primary cultures of eel pituitary cells were performed to investigate the direct effects of kisspeptin on pituitary hormone expression. Human/lamprey kisspeptin exerted a time- and dose-dependent inhibitory effect on LHß expression. All other tested kisspeptins had a similar inhibitory effect on LHß expression. The inhibitory effect of kisspeptins was exerted specifically on LHß as no change was induced on the expression of other glycoprotein hormone subunits (GPα, FSHß and TSHß) nor of growth hormone. These data provide the first evidence for the existence, in the European eel, of a kisspeptin system, which may play a direct inhibitory role on pituitary LHß expression.

Dental Emergencies Management in COVID-19 Pandemic Peak: A Cohort Study.

Due to the global coronavirus disease 2019 pandemic, the high risk of cross-contamination and the overload of hospital facilities have resulted in a real urgency for restricting dental emergency patient flow. In this context, the objectives of the current work were to 1) measure the ability of a triage-based management strategy to limit patient admission and 2) evaluate the success rate of both on-site and remote emergency management regarding symptom relief and pain control over a 1-mo period. We included all patients contacting the dental medicine department for an emergency consultation during the lockdown, between April 1 and April 30, 2020 (N = 570). Following a telephone consultation and based on preestablished admission guidelines, a decision was made at baseline (T0) to either admit the patient for treatment or perform remote management by providing advice and/or drug prescription. Patients were then followed up systematically at 1 wk and 1 mo. Management failure was defined as the need for emergency admission for patients managed remotely since T0 and for new emergency admission for those admitted at T0. The global follow-up rate of patients with a complete data set was 91.4% (N = 521). Of included patients, 49.3% could be managed without admission for emergency reasons for 1 mo. The proportion of successful management was 71.8% and 90.2% at 1 mo for remote and on-site management, respectively. To conclude, the proposed triage-based emergency management strategy with systematic follow-up was a good compromise between limiting patient admission and ensuring effective symptom relief and pain control. The strategy can be useful in situations where regulation of the emergency patient flow is required.

Investigating filler morphology and mechanical properties of new low-shrinkage resin composite types.

Three types of low-shrinkage composites are today commercially available: Ormocers, cationic ring-opening curing systems and highly filled methacrylate-based materials, which cure via free-radical polymerization mechanisms. The aim of this study was to characterize the inorganic fraction of materials belonging to each type and to compare their mechanical properties. Two Ormocers (Admira and an experimental Ormocer V35694), one ring-opening composite (Filtek Silorane) and five methacrylate-based composites [Filtek Supreme XT, Tetric EvoCeram, Grandio, Synergy D6 (Coltène-Whaledent, Langenau, Germany) and an experimental material, V34930] were tested. Inorganic fillers were quantified by thermogravimetric analysis and morphologically characterized by scanning electron microscopy. Dynamic modulus was determined by an impulse excitation technique, static elastic moduli and flexural strength by a three-point bending method. The results were analyzed using ANOVA tests (P < 0.05) and linear correlations. Grandio, V34930 and V35694 exhibited significantly higher filler mass fractions. Both dynamic and static moduli of Grandio and V34930 were significantly higher than the other materials (P < 0.05), although no significant difference in flexural strength was observed between material type (P > 0.05). From the present findings, it was suggested that V35694 and Filtek Silorane exhibit comparable properties to conventional methacrylate-based composites, although clinically the cavity type and location must guide material choice. Under high occlusal load, the use of Grandio and V34930 might be favoured. For small cavities, alternative technologies could be preferred as the need for mechanical resistance is lower and the potential for stress generation is greater.

Core build-up resin composites: an in-vitro comparative study.

AIM: Resin composite (RC) are commonly used under full crowns. However, independent information is lacking to guide practitioners regarding core RC material selection. This study aimed at comparing the flexural properties of a large selection of commercially-available core build-up RCs (CBU-RC), either light-, self- or dual-cure, to conventional light-cure RCs. METHODS: RCs were injected into a 25 × 2×2mm Teflon mold, and either light-cured during 20 s (materials with claimed light-cure characteristics) or covered by aluminum during 10 min (dual- and self-cure CBU-RCs). They were subjected after a one-week water storage at 37.5 °C to three-point bending, and Flexural modulus (E flex) and Flexural Strength (σ f) were calculated (n = 20). Thermogravimetric analysis (n = 3) was performed to determine inorganic filler content (%). RESULTS: For dual-cure CBU-RCs, both RC (p < .0001) and light-curing (p = .0007) had a significant influence on E flex, while only RC was significant for σ f (p < .0001). Between all conventional RCs and CBU-RCs, significant differences were observed (p < .0001), both regarding E flex and σ f, with values ranging from 3.9 to 15.5 GPa and from 76 to 130.3 MPa, respectively. Higher E flex values were observed for light-cure RCs than for self- and dual-cure ones, while no clear trend was noticed regarding σ f. Good linear correlation was found between inorganic filler content and E flex (R 2=0.85, p < .0001), but not with σ f (R 2=0.08, p = .1609). CONCLUSION: This work demonstrated a positive influence of light-curing on dual-cure CBU-RC's E flex. It also highlighted large differences in flexural properties (especially E flex) among the investigated materials, questioning the use of some CBU-RCs as dentin replacement in case of large tissue loss.

Investigating the limits of resin-based luting composite photopolymerization through various thicknesses of indirect restorative materials.

OBJECTIVE: To determine the limitations of using light-curable resin-based luting composites (RBLCs) to bond indirect ceramic/resin-composite restorations by measuring light transmittance through indirect restorative materials and the resulting degree of conversion (DC) of the luting-composites placed underneath. METHODS: Various thicknesses (0-4mm) and shades of LAVA Zirconia and LAVA Ultimate were prepared and used as light curing filters. A commercial, light curable RBLC, RelyX Veneer (control) was compared with four experimental RBLCs of the following composition: TEGDMA/BisGMA (50/50 or 30/70wt%, respectively); camphorquinone/amine (0.2/0.8wt%) or Lucirin-TPO (0.42wt%); microfillers (55wt%) and nanofillers (10wt%). RBLCs covered with the LAVA filter were light-cured for 40s, either with the dual-peak BluephaseG2 or an experimental device emitting either in the blue or violet visible band. The samples were analyzed by Raman spectroscopy to determine DC. Light transmittance through the filters was measured using a common spectroscopy technique. RESULTS: All the factors studied significantly influenced DC (p<0.05). RBLCs with increased TEGDMA content exhibited higher DC. Only small differences were observed comparing DC without filters and filters ≤1mm (p>0.05). For thicknesses ≥2mm, significant reductions in DC were observed (p<0.05). Transmittance values revealed higher filter absorption at 400nm than 470nm. A minimal threshold of irradiance measured through the filters that maintained optimal DC following 40s irradiation was identified for each RBLC formulation, and ranged between 250-500mW/cm2. SIGNIFICANCE: This work confirmed that optimal photopolymerization of RBLCs through indirect restorative materials (≤4mm) and irradiation time of 40s is possible, but only in some specific conditions. The determination of such conditions is likely to be key to clinical success, and all the factors need to be optimized accordingly.

A selenoprotein T-derived peptide protects the heart against ischaemia/reperfusion injury through inhibition of apoptosis and oxidative stress.

AIM: Selenoprotein T (SelT or SELENOT) is a novel thioredoxin-like enzyme whose genetic ablation in mice results in early embryonic lethality. SelT exerts an essential cytoprotective action during development and after injury through its redox-active catalytic site. This study aimed to determine the expression and regulation of SelT in the mammalian heart in normal and pathological conditions and to evaluate the cardioprotective effect of a SelT-derived peptide, SelT43-52(PSELT) encompassing the redox motif which is key to its function, against ischaemia/reperfusion(I/R) injury. METHODS: We used the isolated Langendorff rat heart model and different analyses by immunohistochemistry, Western blot and ELISA. RESULTS: We found that SelT expression is very abundant in embryo but is undetectable in adult heart. However, SelT expression was tremendously increased after I/R. PSELT (5 nmol/L) was able to induce pharmacological post-conditioning cardioprotection as evidenced by a significant recovery of contractility (dLVP) and reduction of infarct size (IS), without changes in cardiac contracture (LVEDP). In contrast, a control peptide lacking the redox site did not confer cardioprotection. Immunoblot analysis showed that PSELT-dependent cardioprotection is accompanied by a significant increase in phosphorylated Akt, Erk-1/2 and Gsk3α-ß, and a decrement of p38MAPK. PSELT inhibited the pro-apoptotic factors Bax, caspase 3 and cytochrome c and stimulated the anti-apoptotic factor Bcl-2. Furthermore, PSELT significantly reduced several markers of I/R-induced oxidative and nitrosative stress. CONCLUSION: These results unravel the role of SelT as a cardiac modulator and identify PSELT as an effective pharmacological post-conditioning agent able to protect the heart after ischaemic injury.

Functional differences between NPFF1 and NPFF2 receptor coupling: high intrinsic activities of RFamide-related peptides on stimulation of [35S]GTPgammaS binding.

By using an optimized [(35)S]GTPgammaS binding assay, the functional activities (potency and efficacy) of peptides belonging to three members of the RFamide family; Neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and 26RFamide, were investigated on NPFF(1) and NPFF(2) receptors stably expressed in Chinese Hamster Ovary (CHO) cells. Despite their large differences in affinity and selectivity, all analogues tested behaved as agonists toward NPFF(1) and NPFF(2) receptors. High NaCl concentration in the assay strongly increased the efficacy toward NPFF(2) receptors and augmented differences among agonists. In low sodium conditions, whereas the potencies of agonists correlated with their affinities for NPFF(1) receptors, NPFF(2) receptors exhibited an extraordinary activity since all compounds tested displayed EC(50) values of GTPgammaS binding lower than their K(I) values. Comparisons of functional values between NPFF(1) and NPFF(2) receptors revealed unexpected potent selective NPFF(2) agonists especially for the PLRFamide and the VGRFamide sequences. By using blocker peptides, we also show that Galpha(i3) and Galpha(s) are the main transducers of NPFF(1) receptors while NPFF(2) are probably coupled with Galpha(i2), Galpha(i3), Galpha(o) and Galpha(s) proteins. Our data indicate that NPPF(1) and NPFF(2) receptors are differently coupled to G proteins in CHO cells.

Effect of the diazepam-binding inhibitor-derived peptide, octadecaneuropeptide, on food intake in goldfish.

An endogenous ligand of central-type benzodiazepine receptors (CBR), the endozepine octadecaneuropeptide (ODN), is a very potent inhibitor of food intake in rodents. Although endozepines have been localized and characterized in the trout hypothalamus, so far, the action of these neuropeptides on feeding behavior has never been investigated in fish. In the present study, we have examined the effect of i.c.v. administration of synthetic rat ODN, its C-terminal octapeptide (OP) and the head-to-tail cyclic analog cyclo(1-8)OP (cOP) on feeding behavior in the goldfish model. i.c.v. injection of graded doses of ODN (2.5-10 pmol/g body weight (BW)) induced a dose-dependent inhibition of food intake, a significant decrease in cumulative food intake during the 60-min period after feeding being observed at doses of 5 and 10 pmol/g BW. The inhibitory effect of a 10 pmol/g BW dose of ODN on food consumption (-39%) was mimicked by an equimolar dose of OP (-42%) and cOP (-53%). The food intake-suppressing activity of ODN (10 pmol/g BW) was not affected by pre-injection of the CBR antagonist flumazenil (200 pmol/g BW). In contrast, the anorexigenic effect of ODN (10 pmol/g BW) was totally suppressed by a selective antagonist of metabotropic endozepine receptors, cyclo(1-8)[dLeu(5)]OP. These data indicate that, in goldfish as in rodents, ODN is a potent inhibitor of food consumption, and that the anorexigenic effect of ODN is not mediated through CBR but through the metabotropic endozepine receptor.

A potential role for the secretogranin II-derived peptide EM66 in the hypothalamic regulation of feeding behaviour.

EM66 is a conserved 66-amino acid peptide derived from secretogranin II (SgII), a member of the granin protein family. EM66 is widely distributed in secretory granules of endocrine and neuroendocrine cells, as well as in hypothalamic neurones. Although EM66 is abundant in the hypothalamus, its physiological function remains to be determined. The present study aimed to investigate a possible involvement of EM66 in the hypothalamic regulation of feeding behaviour. We show that i.c.v. administration of EM66 induces a drastic dose-dependent inhibition of food intake in mice deprived of food for 18 hours, which is associated with an increase of hypothalamic pro-opiomelanocortin (POMC) and melanocortin-3 receptor mRNA levels and c-Fos immunoreactivity in the POMC neurones of the arcuate nucleus. By contrast, i.c.v. injection of EM66 does not alter the hypothalamic expression of neuropeptide Y (NPY), or that of its Y1 and Y5 receptors. A 3-month high-fat diet (HFD) leads to an important decrease of POMC and SgII mRNA levels in the hypothalamus, whereas NPY gene expression is not affected. Finally, we show that a 48 hours of fasting in HFD mice decreases the expression of POMC and SgII mRNA, which is not observed in mice fed a standard chow. Taken together, the present findings support the view that EM66 is a novel anorexigenic neuropeptide regulating hypothalamic feeding behaviour, at least in part, by activating the POMC neurones of the arcuate nucleus.

Cardiovascular effects of native and non-native urotensin II and urotensin II-related peptide on rat and salmon hearts.

Urotensin II (UII) was first discovered in the urophyses of goby fish and later identified in mammals, while urotensin II-related peptide (URP) was recently isolated from rat brain. We studied the effects of UII on isolated heart preparations of Chinook salmon and Sprague-Dawley rats. Native rat UII caused potent and sustained, dose-dependent dilation of the coronary arteries in the rat, whereas non-native UII (human and trout UII) showed attenuated vasodilation. Rat URP dilated rat coronary arteries, with 10-fold less potency compared with rUII. In salmon, native trout UII caused sustained dilation of the coronary arteries, while rat UII and URP caused significant constriction. Nomega-nitro-(l)-arginine methyl (l-NAME) and indomethacin significantly attenuated the URP and rat UII-induced vasodilation in the rat heart. We conclude that UII is a coronary vasodilator, an action that is species form specific. We also provide the first evidence for cardiac actions of URP, possibly via mechanisms common with UII.

Molecular cloning and functional characterization of a type-I neurotensin receptor (NTR) and a novel NTR from the bullfrog brain.

Neurotensin (NT) is a tridecapeptide that functions as a neurotransmitter and neuromodulator in the nervous system. To date, three different types of NT receptor (NTR), NTR1, NTR2 and NTR3, have been identified only in mammalian species. In the present study we isolated the cDNAs for an NTR1 and a novel NTR in the bullfrog brain, designated bfNTR1 and bfNTR4 respectively. bfNTR1 and bfNTR4 encode 422- and 399-amino acid residue proteins respectively. bfNTR1 has a 64% amino acid identity with mammalian NTR1, and 34-37% identity with mammalian NTR2. bfNTR4 exhibits 43% and 45-47% identity with mammalian NTR1 and NTR2 respectively. Both receptors are mainly expressed in the brain and pituitary. bfNTR1 triggers both CRE-luc, a protein kinase A (PKA)-specific reporter, and c-fos-luc, a PKC-specific reporter, activities, indicating that bfNTR1 can activate PKA- and PKC-linked signaling pathways. However, bfNTR4 appears to be preferentially coupled to the PKA-linked pathway as it induces a higher CRE-luc activity than c-fos-luc activity. bfNTRs exhibit different pharmacological properties as compared with mammalian NTRs. Mammalian NTR1 but not NTR2 responds to NT, whereas both bfNTR1 and bfNTR4 show a high sensitivity to NT. SR 48692 and SR 142948A, antagonists for mammalian NTR1 but agonists for mammalian NTR2, function as antagonists for both bfNTR1 and bfNTR4. In conclusion, this report provides the first molecular, pharmacological and functional characterization of two NTRs in a non-mammalian vertebrate. These data should help to elucidate the phylogenetic history of the G protein-coupled NTRs in the vertebrate lineage as well as the structural features that determine their pharmacological properties.

In vivo action of a new octadecaneuropeptide antagonist on neuropeptide Y and corticotropin-releasing hormone mRNA levels in rat.

It has been reported that several of the effects induced by an octadecaneuropeptide (ODN), derived from an 86-amino-acid polypeptide termed diazepam-binding inhibitor, could be mediated by activation of a metabotropic receptor. In order to investigate the role and mechanism of action of ODN in the regulation of corticotropin-releasing factor (CRH) and neuropeptide Y (NPY) expression in the paraventricular nucleus and arcuate nucleus, respectively, we studied the effects of the acute intracerebroventricular administration of ODN (2 microg/rat) and the ODN antagonist to metabotropic receptor, cyclo(1-8)[Dleu5]OP (20 microg/rat), on the gene expression of the two neuropeptides in castrated male rat. ODN administration resulted in a 45% increase in CRH mRNA expression, an effect which was reversed by cyclo(1-8)[Dleu5]OP. When cyclo(1-8)[Dleu5]OP was administered alone, it induced a 19% decrease in CRH mRNA levels. ODN administration induced a 17% decrease in NPY mRNA expression while cyclo(1-8)[Dleu5]OP increased by 21% the hybridization signal. The administration of both ODN and ODN antagonist completely abolished the depressing effect of ODN on NPY mRNA. These data suggest that the effects of ODN on CRH and NPY mRNA might be mediated by interaction with metabotropic receptors. Moreover, since cyclo(1-8)[Dleu5]OP can by itself influence the expression of two peptide mRNAs, it might be suggested that ODN is exerting a tonic influence on NPY and CRH neurons.

Time-course effects of centrally administered native urotensin-II on motor and cardioventilatory activity in trout.

Although in most vertebrate species urotensin-II (UII) is synthesized in neurons of the central nervous system, little is known regarding the physiological actions of UII in the brain. We have investigated the effects of intracerebroventricular (ICV) administration of synthetic trout UII (1, 5, and 50 pmol) on total motor activity (ACT), ventilatory frequency (VF), ventilatory amplitude (VA), and heart rate (HR) in the unanesthetized trout. ICV injection of UII increased ACT in a dose-dependent manner, and the maximal effect was observed at a dose of 5 pmol. At doses of 1 and 5 pmol, UII did not affect VF, VA, or HR. At the highest dose tested (50 pmol), UII not only increased ACT, but also significantly activated VF, VA, and HR. In contrast, ICV injection of synthetic trout angiotensin-II (5 pmol) did not produce any effect on ACT, VF, or VA, but sharply increased HR. These data provide the first evidence that UII can act centrally to induce motor activity in a nonmammalian vertebrate species.