FIRST DATA ON TETRODOTOXINS IN CEPHALOTHRIX MOKIEVSKII (PALAEONEMERTEA, NEMERTEA) AND POSSIBLE INVOLVEMENT OF POISONOUS NEMERTEANS IN TOXIFICATION OF MARINE BIORESOURCES.

We for the first time have recorded the presence of TTX and 11 of its analogues (TTXs) and determined the profile of these toxins by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in Cephalothrix mokievskii, collected off the coast of Sakhalin Island and also revealed differences in the TTXs profile between C. mokievskii and sibling Cephalothrix cf, simula. We discuss the features of the TTXs profile in C. mokievskii, geographical distribution of TTX-containing nemerteans and its possible implication for toxification of marine bioresources.

Investigating Non-Native Ribbon Worm Cephalothrix simula as a Potential Source of Tetrodotoxin in British Bivalve Shellfish.

Tetrodotoxin (TTX) is a potent marine neurotoxin found in several phylogenetically diverse organisms, some of which are sought as seafood. Since 2015, TTX has been reported in bivalve shellfish from several estuarine locations along the Mediterranean and European Atlantic coasts, posing an emerging food safety concern. Although reports on spatial and temporal distribution have increased in recent years, processes leading to TTX accumulation in European bivalves are yet to be described. Here, we explored the hypothesis that the ribbon worm species Cephalothrix simula, known to contain high levels of TTX, could play a role in the trophic transfer of the toxin into shellfish. During a field study at a single location in southern England, we confirmed C. simula DNA in seawater adjacent to trestle-farmed Pacific oysters Magallana gigas (formerly Crassostrea gigas) with a history of TTX occurrence. C. simula DNA in seawater was significantly higher in June and July during the active phase of toxin accumulation compared to periods of either no or continually decreasing TTX concentrations in M. gigas. In addition, C. simula DNA was detected in oyster digestive glands collected on 15 June 2021, the day with the highest recorded C. simula DNA abundance in seawater. These findings show evidence of a relationship between C. simula and TTX occurrence, providing support for the hypothesis that bivalves may acquire TTX through filter-feeding on microscopic life forms of C. simula present in the water column at particular periods each year. Although further evidence is needed to confirm such feeding activity, this study significantly contributes to discussions about the biological source of TTX in European bivalve shellfish.

Epidermal distribution of tetrodotoxin-rich cells in newly hatched larvae of Takifugu spp.

Pufferfish of the genus Takifugu possess tetrodotoxin (TTX), known as "pufferfish toxin" and it is believed that pufferfish eggs and newly hatched larvae utilize TTX as a defensive substance against predators. However, the mechanism for the placement of TTX to specific cells on the larval body surface during the developmental process remains unknown. In this study, we clarify the distribution and characteristics of TTX-rich cells. We performed whole-mount immunohistochemistry (IHC) using anti-TTX monoclonal antibody on larvae of two pufferfish species, Takifugu rubripes and Takifugu alboplumbeus, just after hatching. This allowed observation of the TTX location and compared it with those of wheat germ agglutinin (WGA)-positive (periodic acid-Schiff (PAS)-positive) cells for mucous cells and IHC using anti-Na+/K+-ATPase (NKA) monoclonal antibody for ionocytes. As a result, uniformly scattered localization of TTX-rich cells was commonly observed in the epidermis of the larvae of the two Takifugu species. TTX-rich cells were WGA-negative (PAS-negative) and structurally distinct from NKA-positive cells, suggesting that TTX-rich cells are unreported small cells unique to pufferfish skin, but not mucous cells nor ionocytes.

Electrochemical molecularly imprinted polymer sensor for simple and fast analysis of tetrodotoxin in seafood.

Tetrodotoxin (TTX) is a marine biotoxin whose biosynthesis is associated with the pufferfish. Its distribution is primarily focused in Asian and tropical marine areas. Currently, this group of toxins is classified as emerging in Europe, and its presence could be related to climate change. This incidence has prompted the European Union, with the European Food Safety Authority, to establish control and monitoring mechanisms for TTX in marine products in Europe. In this context, the development of analytical tools capable of ensuring the safety of food products, especially seafood and fish, is a crucial task. This study describes the development of a molecularly imprinted polymer (MIP) based electrochemical sensor for the analysis of TTX. The MIP was synthesized through the electropolymerization of a functional monomer, ortho-phenylenediamine in the presence of a dummy template, voglibose. The MIP sensor was constructed on a screen-printed gold electrode and characterized by cyclic voltammetry. Differential pulse voltammetry, using a redox probe ([Fe(CN)6]3-/4-), was used in the analysis protocol. The developed sensor exhibited a linear response between 5.0 µg mL-1 and 25.0 µg mL-1, with a limit of detection of 1.14 µg mL-1. Its high imprinting efficiency conferred outstanding selectivity towards TTX. The sensor's applicability was confirmed through recovery assays on spiked mussel samples, achieving recoveries of 81.0 %, 110.2 %, and 102.5 % for external standard addition at 30.0, 44.0, and 60.0 µg kg-1, respectively, with relative standard deviations below 15 %. These results are comparable to those obtained using Hydrophilic Interaction Liquid Chromatography coupled with Tandem Mass Spectrometry, a validated method carried out by the European Reference Laboratory for Marine Biotoxins. Thus, the MIP sensor represents a portable, simple, and fast tool with essential analytical functionalities for the sampling phase and pre-selection of laboratory samples for analysis.

Tetrodotoxin and the state-of-the-art progress of its associated analytical methods.

Tetrodotoxin (TTX), which is found in various marine organisms, including pufferfish, shellfish, shrimp, crab, marine gastropods, and gobies, is an effective marine toxin and the cause of many seafood poisoning incidents. Owing to its toxicity and threat to public health, the development of simple, rapid, and efficient analytical methods to detect TTX in various food matrices has garnered increasing interest worldwide. Herein, we reviewed the structure and properties, origin and sources, toxicity and poisoning, and relevant legislative measures of TTX. Additionally, we have mainly reviewed the state-of-the-art progress of analytical methods for TTX detection in the past five years, such as bioassays, immunoassays, instrumental analysis, and biosensors, and summarized their advantages and limitations. Furthermore, this review provides an in-depth discussion of the most advanced biosensors, including cell-based biosensors, immunosensors, and aptasensors. Overall, this study provides useful insights into the future development and wide application of biosensors for TTX detection.

First record of paralytic shellfish toxins in marine pufferfish from the Spanish Mediterranean coast using cell-based assay, automated patch clamp and HPLC-FLD.

Pufferfish is one of the most poisonous marine organisms, responsible for numerous poisoning incidents and some human fatalities due to its capability to accumulate potent neurotoxins such as tetrodotoxins (TTXs) and paralytic shellfish toxins (PSTs). In this study, tissue extracts (muscle, skin, liver, intestinal tract and gonads) obtained from sixteen pufferfish specimens of the Lagocephalus lagocephalus and Sphoeroides pachygaster species, collected along the Spanish Mediterranean coast, were analysed for the presence of voltage-gated sodium channel (also known as Nav channel) blockers using cell-based assay (CBA) and automated patch clamp (APC). No toxicity was observed in any of the S. pachygaster specimens, but toxicity was detected in the liver of most L. lagocephalus specimens. Instrumental analysis of these specimens, as well as in one Lagocephalus sceleratus specimen, by high-performance liquid chromatography coupled to fluorescence detection (HPLC-FLD) was performed, which confirmed the presence of PSTs only in L. lagocephalus specimens. This analysis reported the presence of saxitoxin (STX) and decarbamoylsaxitoxin (dcSTX) in all positive samples, being dcSTX the major analogue. These results demonstrate the ability of this species to accumulate PSTs, being the first report of the presence of PSTs in Mediterranean L.lagocephalus specimens. Furthermore, the presence of high PSTs contents in all five tested tissues of one L. lagocephalus specimen pointed the risk that the presence of this toxic fish in the Mediterranean Sea may represent for seafood safety and human health in case of accidental consumption.

Recent research progress in tetrodotoxin detection and quantitative analysis methods.

Tetrodotoxin (TTX) is a highly potent and widely distributed ion-channel marine neurotoxin; it has no specific antidote and poses a great risk to human health. Therefore, detecting and quantifying TTX to effectively implement prevention strategies is important for food safety. The development of novel and highly sensitive, highly specific, rapid, and simple techniques for trace TTX detection has attracted widespread attention. This review summarizes the latest advances in the detection and quantitative analysis of TTX, covering detection methods based on biological and cellular sensors, immunoassays and immunosensors, aptamers, and liquid chromatography-mass spectrometry. It further discusses the advantages and applications of various detection technologies developed for TTX and focuses on the frontier areas and development directions of TTX detection, providing relevant information for further investigations.

Rattlesnake Crotalphine Analgesic Active on Tetrodotoxin-Sensitive Na+ Current in Mouse Dorsal Root Ganglion Neurons.

Crotalphine is an analgesic peptide identified from the venom of the South American rattlesnake Crotalus durissus terrificus. Although its antinociceptive effect is well documented, its direct mechanisms of action are still unclear. The aim of the present work was to study the action of the crotalid peptide on the NaV1.7 channel subtype, a genetically validated pain target. To this purpose, the effects of crotalphine were evaluated on the NaV1.7 component of the tetrodotoxin-sensitive Na+ current in the dorsal root ganglion neurons of adult mice, using the whole-cell patch-clamp configuration, and on cell viability, using propidium iodide fluorescence and trypan blue assays. The results show that 18.7 µM of peptide inhibited 50% of the Na+ current. The blocking effect occurred without any marked change in the current activation and inactivation kinetics, but it was more important as the membrane potential was more positive. In addition, crotalphine induced an increase in the leakage current amplitude of approximately 150% and led to a maximal 31% decrease in cell viability at a high 50 µM concentration. Taken together, these results point out, for the first time, the effectiveness of crotalphine in acting on the NaV1.7 channel subtype, which may be an additional target contributing to the peptide analgesic properties and, also, although less efficiently, on a second cell plasma membrane component, leading to cell loss.

Is tetrodotoxin intoxication the cause of "zombi voice" in Haiti?

OBJECTIVE: Zombification, a magical and religious process in Haiti, has been scientifically studied and remains relevant. Originating from the convergence of African, Caribbean, and Christian rites, it involves a comatose trance, transforming individuals into living dead through Voodoo practices. Haitian zombies consistently exhibit a preserved expression marked by a nasal voice, a result of nasalization-using nasal cavities as resonators during phonation. The aim of this study was to ascertain the mechanisms through which zombification could impact the voices of the subjects. METHODS: A comprehensive investigation was conducted using both primary and secondary sources. Primary sources involved direct or reported testimonies of individuals undergoing zombification, with audio or video recordings available from the collections of the Laboratory of Anthropology, Archaeology, and Biology (UVSQ/Paris-Saclay University), as well as on the internet. Secondary sources encompassed the entirety of existing literature regarding zombification in Haiti on one hand, alterations in the voices of subjects when mentioned on the other hand, and toxicological hypotheses or evidence available on PubMed/Medline and Google Scholar. RESULTS: Few post-zombification observations exist, but 20th-century studies clarified the physio pathological process, confirming its reality. Wade Davis demonstrated in 1983 that zombification results from poisoning, with effects ranging from reversible to fatal, implicating substances like tetrodotoxin and datura. Nasalization can be natural or pathological, affecting various phonemes. No mutilating acts or surgery have been reported related to Haitian zombification. CONCLUSION: The pharmacological characteristics of tetrodotoxin, coupled with testimonials, present a medical hypothesis elucidating the biological mechanism underlying nasalization in this context. Given that tetrodotoxin induces flaccid paralysis as a neurotropic poison, its neurological impact could account for soft palate paralysis or spasms. Additionally, the severe hypotension induced by tetrodotoxin may elucidate oral and pharyngeal necrosis.

Toxicity Equivalency Factors for Tetrodotoxin Analogues Determined with Automated Patch Clamp on Voltage-Gated Sodium Channels in Neuro-2a Cells.

Tetrodotoxin (TTX) is a potent marine neurotoxin, responsible for numerous poisoning incidents and some human fatalities. To date, more than 30 TTX analogues have been identified, but their individual toxicities and roles in poisoning remain largely unknown. In this work, the toxicity equivalency factors (TEFs) of five TTX analogues were determined by assessing the blockade of voltage-gated sodium channels in Neuro-2a cells using automated patch clamp (APC). All TTX analogues were less toxic than TTX. The derived TEFs were applied to the individual TTX analogues concentrations measured in pufferfish samples, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A comparison of these results with those obtained from APC analysis demonstrated that TEFs can be effectively used to translate LC-MS/MS analytical data into meaningful toxicological information. This is the first study to utilize APC device for the toxicological assessment of TTX analogues, highlighting its potential as a bioanalytical tool for seafood safety management and human health protection.

Determination of tetrodotoxin in human plasma and urine using online MCX SPE column cleanup coupled with liquid chromatography-tandem mass spectrometry.

An efficient technique for quantitative analysis of tetrodotoxin (TTX) in human plasma and urine has been developed, which combines liquid chromatography-tandem mass spectrometry (LC-MS/MS) with online MCX solid phase extraction (SPE) cleanup. Sample preparation, including extraction with acetonitrile containing 0.5 % acetate acid, centrifugation, and filtration, was followed by online SPE cleanup. The whole run-time was less than 15 min, including online cleanup, chromatographic separation, and re-equilibration of the online SPE - LC-MS/MS system. The parameters of sample extraction, purification, separation, and detection were optimized. The matrix-matched internal standard calibration standard curves with linear regression coefficients larger than 0.9990 were established for quantification. The LOD and LOQ for this approach were determined to be 0.1 ng/mL and 0.3 ng/mL, respectively. The recoveries for varied concentrations of TTX in human plasma and urine were 84.9-104.2 % and 89.2-109.6 %, respectively. The matrix effects of TTX in human plasma and urine matrices were 85.5 % and 74.3 %, respectively, and both the inter- and intra-day precision values were less than 9.5 %. This analytical method was successfully employed for detecting TTX in biological samples from a poisoned patient who accidentally ingested the nassarius glans.

Tetrodotoxin-resistant mechanosensitivity and L-type calcium channel-mediated spontaneous calcium activity in enteric neurons.

Gut motility undergoes a switch from myogenic to neurogenic control in late embryonic development. Here, we report on the electrical events that underlie this transition in the enteric nervous system, using the GCaMP6f reporter in neural crest cell derivatives. We found that spontaneous calcium activity is tetrodotoxin (TTX) resistant at stage E11.5, but not at E18.5. Motility at E18.5 was characterized by periodic, alternating high- and low-frequency contractions of the circular smooth muscle; this frequency modulation was inhibited by TTX. Calcium imaging at the neurogenic-motility stages E18.5-P3 showed that CaV1.2-positive neurons exhibited spontaneous calcium activity, which was inhibited by nicardipine and 2-aminoethoxydiphenyl borate (2-APB). Our protocol locally prevented muscle tone relaxation, arguing for a direct effect of nicardipine on enteric neurons, rather than indirectly by its relaxing effect on muscle. We demonstrated that the ENS was mechanosensitive from early stages on (E14.5) and that this behaviour was TTX and 2-APB resistant. We extended our results on L-type channel-dependent spontaneous activity and TTX-resistant mechanosensitivity to the adult colon. Our results shed light on the critical transition from myogenic to neurogenic motility in the developing gut, as well as on the intriguing pathways mediating electro-mechanical sensitivity in the enteric nervous system. HIGHLIGHTS: What is the central question of this study? What are the first neural electric events underlying the transition from myogenic to neurogenic motility in the developing gut, what channels do they depend on, and does the enteric nervous system already exhibit mechanosensitivity? What is the main finding and its importance? ENS calcium activity is sensitive to tetrodotoxin at stage E18.5 but not E11.5. Spontaneous electric activity at fetal and adult stages is crucially dependent on L-type calcium channels and IP3R receptors, and the enteric nervous system exhibits a tetrodotoxin-resistant mechanosensitive response. Abstract figure legend Tetrodotoxin-resistant Ca2+ rise induced by mechanical stimulation in the E18.5 mouse duodenum.

Evolution of ion channels in cetaceans: a natural experiment in the tree of life.

Cetaceans represent a natural experiment within the tree of life in which a lineage changed from terrestrial to aquatic habitats. This shift involved phenotypic modifications, representing an opportunity to explore the genetic bases of phenotypic diversity. Among the different molecular systems that maintain cellular homeostasis, ion channels are crucial for the proper physiological functioning of all living species. This study aims to explore the evolution of ion channels during the evolutionary history of cetaceans. To do so, we created a bioinformatic pipeline to annotate the repertoire of ion channels in the genome of the species included in our sampling. Our main results show that cetaceans have, on average, fewer protein-coding genes and a higher percentage of annotated ion channels than non-cetacean mammals. Signals of positive selection were detected in ion channels related to the heart, locomotion, visual and neurological phenotypes. Interestingly, we predict that the NaV1.5 ion channel of most toothed whales (odontocetes) is sensitive to tetrodotoxin, similar to NaV1.7, given the presence of tyrosine instead of cysteine, in a specific position of the ion channel. Finally, the gene turnover rate of the cetacean crown group is more than three times faster than that of non-cetacean mammals.

The impact of tetrodotoxin (TTX) on the gut microbiome in juvenile tiger pufferfish, Takifugu rubripes.

Tetrodotoxin (TTX) is a potent neurotoxin that accumulates in Takifugu rubripes, commonly known as pufferfish, through the ingestion of TTX-bearing organisms as part of their food chain. Although researchers believe that pufferfish use TTX to relieve stress, data are not currently available on how TTX affects the gut microbiota of pufferfish. To address this gap, our study aimed to investigate whether administering TTX to fish could alter their gut microbiota and overall health under various salinity conditions, including 30.0 ppt, 8.5 ppt, and 1.7 ppt salinity, which represent full-strength, isosmotic, and low-salinity stress, respectively. We analyzed the effect of TTX ingestion on the community structure, core microbiome, and metabolic capabilities of the gut microbiome using high-throughput sequencing technologies. The predominant bacterial taxa within the gut microbiome were Firmicutes (21-85%), Campilobacterota (2.8-67%), Spirochaetota (0.5-14%), and Proteobacteria (0.7-9.8%), with Mycoplasma, uncultured Arcobacteraceae, Brevinema, Vibrio, Rubritalea, and uncultured Pirellulaceae as core genera. Our findings indicated that the impact of TTX on high-abundance genera at 30.0 ppt and 8.5 ppt salinity levels was negligible, indicating their stability and resilience to TTX ingestion. However, at 1.7 ppt, TTX-fed fish showed a significant increase in uncultured Arcobacteraceae. Furthermore, our analysis of TTX-fed fish revealed taxonomic alterations in low-abundance taxa, which altered the predicted functions of the gut microbiota at all salinity levels. These results suggest that TTX administration could cause subtle effects on the metabolic functions of gut microbial communities. Overall, our study provides insights into the complex relationship between a TTX-accumulating animal, T. rubripes, and its gut microbiota.

Tetrodotoxin Derivatization with a Newly Designed Boron Reagent Leads to Conventional Reversed-Phase Liquid Chromatography.

Tetrodotoxin (TTX) is a representative natural toxin causing pufferfish food poisoning, which is especially prominent in East and Southeast Asia, including Japan. TTX has been analyzed through post-column derivatization high-performance liquid chromatography (HPLC), ion-pair LC-MS(/MS), and hydrophilic interaction liquid chromatography (HILIC)-MS(/MS) as alternatives to the mouse bioassay method. However, post-column derivatization requires a system for online derivatization reactions, and with the ion-pair LC-MS approach, it is difficult to remove residual ion-pair reagents remaining in the equipment. Moreover, HILIC-MS provides poor separation compared to reversed-phase (RP) HPLC and requires a long time to reach equilibration. Therefore, we decided to develop a TTX analytical method using pre-column derivatization and RP HPLC for the rapid assessment of outbreak samples, including food remnants. In this study, we focused on the vic-diol moiety of TTX and designed a new derivatization reagent coded as NBD-H-DAB. This NBD-H-DAB was synthesized from 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H) and 3-fluoro-2-formylphenylboronic acid (FFPBA) with a simple reaction system and rapidly converted to its boronate form, coded NBD-H-PBA, in an aqueous reaction solution. The NBD-H-PBA demonstrated appropriate hydrophobicity to be retained on the RP analytical column and successfully detected with a UV spectrometer. It was easily reacted with the vic-diol moiety of TTX (C6 and C11) to synthesized a boronic ester. The derivatized TTX could be detected using the RP HPLC-UV, and the limit of detection in the fish flesh samples was 0.06 mg/kg. This novel pre-column derivatization of TTX with NBD-H-PBA proves capable for the analysis of TTX.

Polymeric Prodrugs using Dynamic Covalent Chemistry for Prolonged Local Anesthesia.

Depot-type drug delivery systems are designed to deliver drugs at an effective rate over an extended period. Minimizing initial "burst" can also be important, especially with drugs causing systemic toxicity. Both goals are challenging with small hydrophilic molecules. The delivery of molecules such as the ultrapotent local anesthetic tetrodotoxin (TTX) exemplifies both challenges. Toxicity can be mitigated by conjugating TTX to polymers with ester bonds, but the slow ester hydrolysis can result in subtherapeutic TTX release. Here, we developed a prodrug strategy, based on dynamic covalent chemistry utilizing a reversible reaction between the diol TTX and phenylboronic acids. These polymeric prodrugs exhibited TTX encapsulation efficiencies exceeding 90 % and the resulting polymeric nanoparticles showed a range of TTX release rates. In vivo injection of the TTX polymeric prodrugs at the sciatic nerve reduced TTX systemic toxicity and produced nerve block lasting 9.7±2.0 h, in comparison to 1.6±0.6 h from free TTX. This approach could also be used to co-deliver the diol dexamethasone, which prolonged nerve block to 21.8±5.1 h. This work emphasized the usefulness of dynamic covalent chemistry for depot-type drug delivery systems with slow and effective drug release kinetics.

Tissue Localization of Tetrodotoxin in the Flatworm Planocera multitentaculata (Platyhelminthes: Polycladida).

Tetrodotoxin (TTX), a pufferfish toxin, is a highly potent neurotoxin that has been found in a wide variety of animals. The TTX-bearing flatworm Planocera multitentaculata possesses a large amount of TTX and is considered responsible for the toxification of TTX-bearing animals such as pufferfish (Takifugu and Chelonodon) and the toxic goby Yongeichthys criniger. However, the mechanism underlying TTX accumulation in flatworms remains unclear. Previous studies have been limited to identifying the distribution of TTX in multiple organs, such as the digestive organs, genital parts, and the remaining tissues of flatworms. Here, we performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and immunohistochemical staining using a monoclonal anti-TTX antibody to elucidate the detailed localization of TTX in the tissues and organs of the flatworm P. multitentaculata. Immunohistochemical staining for P. multitentaculata showed that TTX-specific signals were detected not only in the ovaries and pharynx but also in many other tissues and organs, whereas no signal was detected in the brain, Lang's vesicle, and genitalia. In addition, combined with LC-MS/MS analysis, it was revealed for the first time that TTX accumulates in high concentrations in the basement membrane and epidermis. These findings robustly support the hypotheses of "TTX utilization protection from predators."

Functional effects of drugs and toxins interacting with NaV1.4.

NaV1.4 is a voltage-gated sodium channel subtype that is predominantly expressed in skeletal muscle cells. It is essential for producing action potentials and stimulating muscle contraction, and mutations in NaV1.4 can cause various muscle disorders. The discovery of the cryo-EM structure of NaV1.4 in complex with ß1 has opened new possibilities for designing drugs and toxins that target NaV1.4. In this review, we summarize the current understanding of channelopathies, the binding sites and functions of chemicals including medicine and toxins that interact with NaV1.4. These substances could be considered novel candidate compounds or tools to develop more potent and selective drugs targeting NaV1.4. Therefore, studying NaV1.4 pharmacology is both theoretically and practically meaningful.

Epithelium-derived 6-nitrodopamine modulates noradrenaline-induced contractions in human seminal vesicles.

AIMS: To evaluate the basal release of 6-nitrodopamine (6-ND) from human isolated seminal vesicles (HISV) and to characterize its action and origin. MAIN METHODS: Left HISV obtained from patients undergoing prostatectomy surgery was suspended in a 3-mL organ bath containing warmed (37 °C) and gassed (95%O2:5%CO2) Krebs-Henseleit's solution (KHS) with ascorbic acid. An aliquot of 2 mL of the supernatant was used to quantify catecholamines by LC-MS/MS. For functional studies, concentration-responses curves to catecholamines were obtained, and pEC50 and Emax values were calculated. Detection of tyrosine hydroxylase and S100 protein were also carried out by both immunohistochemistry and fluorescence in-situ hybridization assays (FISH). KEY FINDINGS: Basal release of 6-ND was higher than the other catecholamines (14.76 ± 14.54, 4.99 ± 6.92, 3.72 ± 4.35 and 5.13 ± 5.76 nM for 6-ND, noradrenaline, adrenaline, and dopamine, respectively). In contrast to the other catecholamines, the basal release of 6-ND was not affected by the sodium current (Nav) channel inhibitor tetrodotoxin (1 µM; 10.4 ± 8.9 and 10.4 ± 7.9 nM, before and after tetrodotoxin, respectively). All the catecholamines produced concentration-dependent HISV contractions (pEC50 4.1 ± 0.2, 4.9 ± 0.3, 5.0 ± 0.3, and 3.9 ± 0.8 for 6-ND, noradrenaline, adrenaline, and dopamine, respectively), but 6-ND was 10-times less potent than noradrenaline and adrenaline. However, preincubation with very low concentration of 6-ND (10-8 M, 30 min) produced significant leftward shifts of the concentration-response curves to noradrenaline. Immunohistochemical and FISH assays identified tyrosine hydroxylase in tissue epithelium of HISV strips. SIGNIFICANCE: Epithelium-derived 6-ND is the major catecholamine released from human isolated seminal vesicles and that modulates smooth muscle contractility by potentiating noradrenaline-induced contractions.