Recombinant Scyliorhinus canicula Urotensin-2

What is Scyliorhinus canicula Urotensin-2 and how does the recombinant version differ from native?

Scyliorhinus canicula (dogfish) Urotensin-2 is a vasoactive peptide originally isolated from dogfish that demonstrates potent hypertensive activity. The native peptide is produced in vivo and has been shown to cause sustained and dose-dependent increases in arterial blood pressure and pulse pressure when administered to conscious dogfish . Recombinant Scyliorhinus canicula Urotensin-2 refers to the artificially synthesized version of this peptide, typically produced through bacterial or mammalian expression systems. While the recombinant version maintains the same amino acid sequence and bioactivity as the native peptide, it offers advantages including consistent purity, scalable production, and the ability to introduce specific modifications for research purposes.

The fundamental difference lies in the production method rather than structure or function. Recombinant versions are designed to precisely mimic the native peptide's pharmacological effects, producing similar dose-dependent increases in arterial pressure and vascular muscle contraction as observed in studies with synthetic dogfish urotensin II .

What are the structural characteristics of Scyliorhinus canicula Urotensin-2?

Urotensin-2 peptides are characterized by a cyclic structure formed by a disulfide bridge between cysteine residues, similar to the human U-II which has a sequence of ETPDCFWKYCV with a disulfide bridge between positions 5 and 10 . Though the search results don't provide the exact sequence for Scyliorhinus canicula U-II, structural studies on urotensin-II generally reveal:

• A core region with a highly compact conformation

• Formation of a hydrophobic pocket

• The presence of conserved cyclic region that is critical for biological activity

Nuclear magnetic resonance (NMR) spectroscopy studies of U-II have demonstrated that the peptide adopts a preferential conformation in solution without classical secondary structures . This structural configuration is crucial for receptor binding and subsequent biological activity. The introduction of non-natural amino acids into the sequence, such as replacing Tyr with the bulkier 2-Nal [(2-naphthyl)-L-alanine], has been shown to maintain similar potency in agonist activity while improving receptor binding affinity .

How is Scyliorhinus canicula Urotensin-2 related to urotensin peptides in other species?

Urotensin-II is part of a family of structurally related neuropeptides present in all vertebrates . Evolutionary studies suggest that urotensin peptides and their receptors derive from a single ancestral ligand-receptor pair that existed before the emergence of vertebrates . This ancestral pair duplicated to generate one somatostatin peptide with two receptors and one urotensin-II peptide with one receptor, which subsequently expanded through whole-genome duplications during vertebrate evolution .

The teleost lineage (bony fish) possesses multiple urotensin genes, including UII, URP (Urotensin-related peptide), URP1, and URP2, along with five UTS2R (Urotensin-II receptor) genes . This diversity highlights the evolutionary conservation and importance of the urotensin system across species. Dogfish (Scyliorhinus canicula) U-II represents one specific member of this extended peptide family, with species-specific structural characteristics that influence its biological activity. For instance, studies have shown that while dogfish U-II produces significant hypertensive responses in dogfish, goby (Gillichthys mirabilis) U-II does not elicit significant responses under the same conditions .

What are the optimal concentrations for using recombinant Scyliorhinus canicula Urotensin-2 in cardiovascular research?

Based on experimental data with dogfish urotensin II, the following concentration guidelines can be established for various research applications:

For in vivo studies:

• Effective dose range: 0.1-1.0 nmol for bolus injections

• Optimal dose for maximum response: 0.5 nmol (produces a 38.6 ± 4.2% increase in mean arterial pressure over basal values)

• Comparative reference: 5 nmol of epinephrine produces a 24.8 ± 3.3% rise in mean arterial pressure

For in vitro vascular contractility studies:

• Concentration range: 10^-8 to 10^-5 M

• pD2 value (negative logarithm of EC50): 6.58 ± 0.07

• Maximum contractile force: 1.3 mN at 10^-5 M concentration

When designing experiments, researchers should consider that recombinant versions may exhibit slightly different potency profiles compared to synthetic peptides, necessitating preliminary dose-response studies to establish optimal concentrations for specific experimental setups.

How should I design experiments to study the mechanism of action of recombinant Scyliorhinus canicula Urotensin-2?

To effectively investigate the mechanism of action of recombinant Scyliorhinus canicula Urotensin-2, consider the following experimental approaches based on established protocols:

• Receptor antagonist studies:

  • Pre-treatment with phentolamine (an alpha-adrenergic antagonist) has been shown to completely abolish the hypertensive response to dogfish urotensin II (0.5 nmol) . This indicates involvement of catecholamine release in the mechanism of action. Design similar experiments using specific receptor antagonists to identify the receptor systems involved.

• Isolated tissue preparations:

  • Utilize isolated vascular rings (like the first afferent branchial artery of dogfish) to study direct contractile effects .

  • Test the effects of neural transmission blockers like tetrodotoxin (1 μM) and prostaglandin synthesis inhibitors like indomethacin (14 μM) to determine if the contractile response involves neural mechanisms or prostaglandin-mediated pathways .

• Comparative species studies:

  • Include parallel experiments with urotensin II from different species to identify species-specific effects. For example, goby urotensin II (0.5 nmol) did not elicit significant hypertensive responses in dogfish under conditions where dogfish urotensin II produced marked effects .

• Cellular signaling investigations:

  • Examine intracellular signaling pathways activated by urotensin II, particularly those related to G-protein coupled receptor activation, given that urotensin II receptors (UTS2R) belong to this receptor family .

The experimental design should incorporate appropriate controls, dose-response relationships, and time-course analyses to fully characterize the pharmacological properties of the recombinant peptide.

What methods are recommended for evaluating recombinant Scyliorhinus canicula Urotensin-2 effects on renal function?

Recent research has implicated the urotensin II system in kidney function and chronic kidney disease . To evaluate the effects of recombinant Scyliorhinus canicula Urotensin-2 on renal function, consider these methodological approaches:

• Kidney expression profiling:

  • Quantify UII and URP mRNA expression levels in kidney tissues using qPCR

  • Perform immunostaining to localize expression, noting that UII and URP are predominantly expressed in renal tubules

• Plasma and urine biomarker analysis:

  • Measure plasma UII and URP concentrations (baseline plasma UII concentrations are approximately 2 ng/mL in control mice)

  • Assess renal function parameters, including urine albumin-creatinine ratio (ACR), which increases approximately 3-fold in STZ-treated diabetic mice

• Kidney tissue culture model:

  • Establish primary kidney cell cultures or organ cultures to assess direct effects of recombinant Scyliorhinus canicula Urotensin-2 on renal cells

  • Monitor changes in cell signaling, inflammatory markers, and fibrotic pathways

• Experimental kidney disease models:

  • Utilize streptozotocin (STZ)-induced diabetic models, which demonstrate upregulation of the urotensin system

  • Assess how administration of recombinant Scyliorhinus canicula Urotensin-2 affects disease progression in these models

When designing these experiments, include appropriate dose ranges based on the known potency of urotensin-II (EC50 = 0.1 nM for human urotensin-II) and incorporate time-course analyses to capture both acute and chronic effects on renal function.

How does Scyliorhinus canicula Urotensin-2 compare to human Urotensin-2 in receptor binding and physiological effects?

Understanding the comparative pharmacology of dogfish and human Urotensin-2 is crucial for translational research. These peptides show important similarities and differences:

Receptor Binding Properties:
Human Urotensin-2 is a potent endogenous peptide agonist for the urotensin-II receptor with an EC50 value of 0.1 nM . While specific binding data for Scyliorhinus canicula Urotensin-2 is not directly provided in the search results, comparative studies have shown species-specific differences in receptor interactions. For example, dogfish urotensin II produces significant hypertensive responses in dogfish, while goby urotensin II does not under the same conditions .

Physiological Effects:
Both human and dogfish Urotensin-2 exhibit potent cardiovascular effects, but with species-specific patterns:

• Vasoactive effects: Human U-II displays arterio-selective vasoconstriction and vasodilation in mammals, with effects varying between species . Dogfish U-II demonstrates potent hypertensive activity in dogfish, increasing mean arterial pressure by up to 38.6% over basal values .

• Mechanism differences: Dogfish U-II hypertensive effects are mediated partly through catecholamine release, as phentolamine pre-treatment abolishes the response . Human U-II, in addition to vascular effects, has been shown to mediate bronchoconstriction .

• Structural basis for differences: The varying effects across species likely stem from differences in the peptide sequence and structure, especially in the regions outside the conserved cyclic core. Structure-activity relationship (SAR) studies have shown that modifications to specific amino acids can significantly alter receptor binding and biological activity .

These comparative differences highlight the importance of species-specific research when working with urotensin peptides and extrapolating findings across species barriers.

What structural modifications of recombinant Scyliorhinus canicula Urotensin-2 can enhance its research utility?

Strategic modifications to recombinant Scyliorhinus canicula Urotensin-2 can enhance its research utility through improved stability, altered receptor selectivity, or modified pharmacokinetic properties. Based on structure-activity relationship studies on urotensin-II peptides, several approaches can be considered:

• Amino acid substitutions in the core region:

  • Replacing the Tyr residue with bulkier (2-naphthyl)-L-alanine (2-Nal) has been shown to maintain similar agonist potency while improving receptor binding affinity six-fold in goby U-II .

  • Introduction of non-natural amino acids can provide resistance to enzymatic degradation, extending the peptide's biological half-life.

• Modifications to the cyclic structure:

  • The disulfide bridge between cysteine residues is critical for biological activity, similar to the arrangement in human U-II (positions 5 and 10) .

  • Alternative cyclization strategies, such as lactam bridges or hydrocarbon stapling, could provide enhanced stability while maintaining the critical spatial arrangement of key residues.

• N-terminal and C-terminal modifications:

  • Terminal modifications can protect against exopeptidase degradation, extending in vivo half-life.

  • Addition of polyethylene glycol (PEGylation) can dramatically increase circulation time and reduce immunogenicity.

• Reporter-conjugated variants:

  • Fluorescent or radioactive labeling at non-critical positions can create valuable tools for receptor localization and binding studies.

  • Biotinylation can facilitate pull-down assays to identify novel interaction partners.

When designing modified variants, researchers should systematically evaluate how each modification affects receptor binding, signaling efficacy, and in vivo stability through comparative pharmacological characterization against the unmodified peptide.

What are the challenges in developing selective agonists or antagonists based on Scyliorhinus canicula Urotensin-2 structure?

Developing selective agonists or antagonists based on the Scyliorhinus canicula Urotensin-2 structure presents several significant challenges that researchers must address:

• Receptor subtype selectivity:
  The evolution of the urotensin system has led to multiple receptor subtypes across species. Teleost fish possess five UTS2R genes (UTS2R1-5) , while the receptor landscape in elasmobranchs like dogfish may differ. Designing ligands with selectivity for specific receptor subtypes requires detailed understanding of subtype-specific binding pockets and activation mechanisms.

• Conserved structural elements:
  The core region of U-II adopts a highly compact conformation with a hydrophobic pocket , which is likely essential for receptor recognition. Modifications that maintain this critical structural feature while altering functional outcomes (agonism vs. antagonism) require sophisticated structure-based design approaches.

• Species differences in pharmacology:
  The observation that dogfish U-II elicits significant responses in dogfish while goby U-II does not highlights species-specific pharmacology. Translating findings from one species to another (particularly to humans for therapeutic development) presents considerable challenges in predicting efficacy and safety.

• Multiple signaling pathways:
  U-II receptors likely couple to multiple G-protein signaling pathways, potentially activating distinct downstream effects. Developing biased ligands that selectively activate beneficial pathways while avoiding detrimental ones requires comprehensive understanding of signal transduction mechanisms.

• Pharmacokinetic considerations:
  Peptide-based compounds face inherent challenges including poor oral bioavailability, rapid clearance, and limited blood-brain barrier penetration. Engineering variants with improved drug-like properties while maintaining receptor selectivity and potency presents significant technical hurdles.

Researchers should employ iterative design-test cycles, leveraging computational modeling, medicinal chemistry, and high-throughput screening to overcome these challenges and develop highly selective pharmacological tools.

How can I verify the biological activity of recombinant Scyliorhinus canicula Urotensin-2 preparations?

Verifying the biological activity of recombinant Scyliorhinus canicula Urotensin-2 preparations is crucial for experimental reliability. Based on established methodologies, the following approaches are recommended:

• In vitro vascular contractility assay:

  • Use isolated rings of vascular muscle (preferably from the first afferent branchial artery of dogfish if available)

  • Measure contractile force in response to increasing concentrations of the peptide

  • Compare to reference data: dogfish urotensin II produces dose-dependent contraction with pD2 = 6.58 ± 0.07 and maximum contractile force of 1.3 mN at 10^-5 M

  • If dogfish tissues are unavailable, rat aortic rings or other mammalian vascular preparations can serve as alternative bioassay systems, though with potentially different potency

• Receptor binding assays:

  • Perform competitive binding assays using cell lines expressing the urotensin-II receptor

  • Calculate binding affinity (Ki) and compare to reference standards

  • Human urotensin-II has an EC50 of 0.1 nM at the human urotensin-II receptor , which can serve as a benchmark

• Cell signaling assays:

  • Measure intracellular calcium mobilization or other second messenger responses in urotensin receptor-expressing cells

  • Construct dose-response curves to determine EC50 values

  • Compare to historical data or positive controls run in parallel

• Analytical verification:

  • Confirm peptide integrity through mass spectrometry to verify the expected molecular weight (human U-II has M.Wt of 1388.57)

  • Perform HPLC analysis to assess purity and detect potential degradation products

What factors can influence experimental variability when working with recombinant Scyliorhinus canicula Urotensin-2?

Multiple factors can contribute to experimental variability when working with recombinant Scyliorhinus canicula Urotensin-2, potentially confounding research results. Understanding and controlling these variables is essential for reproducible outcomes:

• Peptide stability and handling:

  • Freeze-thaw cycles: Repeated freezing and thawing can lead to peptide degradation

  • Storage conditions: Optimal storage is in desiccated form at -20°C

  • Solution preparation: Proper reconstitution and dilution techniques are critical for maintaining activity

  • Disulfide bridge integrity: Reducing conditions can disrupt the essential disulfide bridge between cysteine residues

• Experimental model variations:

  • Species differences: The response to urotensin-II varies significantly between species . For instance, dogfish urotensin II elicits hypertensive responses in dogfish, while goby urotensin II does not

  • Tissue preparation variability: For isolated vessel studies, differences in tissue handling and preparation can affect contractile responses

  • Individual animal variability: Genetic and physiological differences between individual animals can impact responses

• Receptor expression and sensitivity:

  • Receptor downregulation: Prior exposure to urotensin-II or related peptides may lead to receptor desensitization

  • Expression levels: Variation in receptor density across tissues or cell cultures can alter response magnitude

  • Post-translational modifications: Differences in receptor glycosylation or phosphorylation states can affect binding and signaling

• Experimental conditions:

  • Buffer composition: pH, ionic strength, and presence of carrier proteins can influence peptide stability and activity

  • Temperature: Enzymatic degradation rates and receptor binding kinetics are temperature-dependent

  • Co-administered agents: Presence of anesthetics (for in vivo studies) or other pharmacological agents may interact with urotensin-II effects

• Technical parameters:

  • Route of administration (for in vivo studies): The cardiovascular effects of dogfish urotensin II have been studied using celiac artery injection

  • Dose calculation errors: Accurate measurement of small peptide quantities is challenging

  • Timing of measurements: The hypertensive response to dogfish urotensin II is sustained , so timing of measurements can influence results

Controlling these variables through standardized protocols, appropriate controls, and sufficient replication is essential for generating reliable and reproducible data.

How is Scyliorhinus canicula Urotensin-2 contributing to understanding evolutionary aspects of neuropeptide systems?

Scyliorhinus canicula (dogfish) Urotensin-2 represents a valuable model for investigating the evolutionary conservation and diversification of neuropeptide systems. Research in this area is revealing important insights into vertebrate neuroendocrine evolution:

• Evolutionary origins of peptide-receptor systems:
  Somatostatin (SS) and urotensin II (UII) families, including their receptors (SSTR and UTS2R), likely derive from a single ancestral ligand-receptor pair that existed before the emergence of vertebrates . This ancestral system duplicated to generate one SS peptide with two receptors and one UII peptide with one receptor, which then expanded through whole-genome duplications during vertebrate evolution .

• Expansion through genome duplications:
  The teleost lineage possesses four UII genes (UII, URP, URP1, and URP2) and five UTS2R genes (UTS2R1-5) , highlighting how the three whole-genome duplications (1R, 2R, and 3R) that occurred during vertebrate evolution shaped neuropeptide diversity. Studying dogfish U-II provides insight into the evolutionary stage before the teleost-specific genome duplication.

• Structural conservation amid functional divergence:
  While the core structure of urotensin peptides is conserved across species, their physiological effects can differ significantly. For example, dogfish U-II produces hypertensive responses in dogfish, while goby U-II does not under the same conditions . This functional divergence despite structural similarity provides a window into how ligand-receptor co-evolution drives species-specific adaptations.

• Comparative physiology insights:
  The varying cardiovascular effects of urotensin peptides across species reflect evolutionary adaptations in cardiovascular regulation. Studying these differences helps understand how environmental pressures and physiological demands shaped neuroendocrine control systems during vertebrate evolution.

Future research combining molecular phylogenetics, comparative physiology, and structural biology approaches will further illuminate how the urotensin system evolved and adapted to diverse physiological roles across the vertebrate lineage.

What are emerging research applications for recombinant Scyliorhinus canicula Urotensin-2 in biomedical science?

Recombinant Scyliorhinus canicula Urotensin-2 is finding increasing applications in biomedical research, particularly in understanding cardiovascular and renal pathophysiology, and in developing novel therapeutic strategies:

• Cardiovascular disease research:
  Dogfish urotensin II demonstrates potent hypertensive activity and causes vascular muscle contraction , making it valuable for studying:

  • Hypertension mechanisms and novel antihypertensive approaches

  • Vascular remodeling processes

  • Comparative cardiovascular pharmacology across species to identify conserved therapeutic targets

• Renal disease models:
  The urotensin II system has been implicated in chronic kidney disease . STZ-induced diabetic mice show upregulation of UII and URP in kidney tissues, predominantly in renal tubules, with increased plasma concentrations . Recombinant Scyliorhinus canicula Urotensin-2 can serve as a tool to investigate:

  • Kidney-specific effects of urotensin II signaling

  • Role in diabetic nephropathy progression

  • Potential for urotensin receptor antagonism as a therapeutic strategy

• Peptide drug development platforms:
  Structure-activity relationship studies on urotensin-II can be enhanced using recombinant Scyliorhinus canicula Urotensin-2 as a template for:

  • Developing selective urotensin receptor modulators

  • Engineering peptides with improved pharmacokinetic properties

  • Creating peptide-based imaging probes for receptor visualization

• Comparative endocrinology:
  The evolutionary relationship between somatostatin and urotensin peptide families makes recombinant Scyliorhinus canicula Urotensin-2 valuable for:

  • Understanding co-evolution of peptide-receptor pairs

  • Investigating conservation of signaling pathways across species

  • Identifying novel receptor subtypes and signaling mechanisms

These emerging applications highlight the value of recombinant Scyliorhinus canicula Urotensin-2 not only as a tool for basic science but also as a potential template for therapeutic development targeting the urotensin system in various disease states.

What are the key unresolved questions about Scyliorhinus canicula Urotensin-2 that warrant further investigation?

Despite significant advances in understanding Urotensin-2 biology, several fundamental questions specific to Scyliorhinus canicula Urotensin-2 remain unresolved and merit dedicated research efforts:

• Receptor subtype selectivity and signaling:

  • Which specific urotensin receptor subtypes does Scyliorhinus canicula Urotensin-2 preferentially activate?

  • Do different receptor subtypes mediate distinct physiological responses?

  • What intracellular signaling pathways are activated by receptor binding, and how do these contribute to observed physiological effects?

• Physiological roles beyond cardiovascular regulation:

  • What is the role of endogenous Scyliorhinus canicula Urotensin-2 in non-cardiovascular systems (e.g., central nervous system, immune system)?

  • How does Scyliorhinus canicula Urotensin-2 interact with other neuroendocrine systems in dogfish?

  • Does it play a role in osmoregulation, metabolism, or stress responses in elasmobranchs?

• Molecular evolution and structure-function relationships:

  • What specific structural features distinguish Scyliorhinus canicula Urotensin-2 from other species' urotensin peptides?

  • How do these structural differences account for the differing physiological effects observed between species?

  • Can evolutionary analysis of urotensin peptides reveal novel insights into receptor binding mechanisms?

• Pathophysiological implications:

  • Is the urotensin system involved in cardiovascular or renal pathologies in elasmobranchs?

  • Could comparative studies between mammals and elasmobranchs reveal protective mechanisms against urotensin-mediated pathologies?

  • How does the role of urotensin in chronic kidney disease in mammals compare to its function in elasmobranch kidney physiology?

• Pharmacological tool development:

  • Can Scyliorhinus canicula Urotensin-2 serve as a template for developing receptor subtype-selective ligands?

  • What modifications to the peptide structure would enhance its utility as a pharmacological probe?

  • Could synthetic analogues based on Scyliorhinus canicula Urotensin-2 provide therapeutic leads for human cardiovascular or renal disease?

Addressing these questions through interdisciplinary approaches combining molecular biology, comparative physiology, structural biology, and pharmacology would significantly advance our understanding of this important peptide system and potentially lead to novel therapeutic strategies.