Synthesis and Fabrication of Surface-Active Microparticles Using a Membrane Emulsion Technique and Conjugation of Model Protein via Strain-Promoted Azide-Alkyne Click Chemistry in Physiological Conditions.

The rapid surface immobilization of protein on monodispersed polyester microcarriers is reported. A model protein, functionalized with a dibenzocyclooctyne core, immobilizes on the surface of azide-terminal polycaprolactone microcarriers within 10 min compared to 12 h for other conjugation techniques, and it is conducted in physiological conditions and in the absence of coupling reagents.

An in vitro investigation into the effects of 10 Hz cyclic loading on tenocyte metabolism.

Tendinopathy is a prevalent, highly debilitating condition, with poorly defined etiology. A wide range of clinical treatments has been proposed, with systematic reviews largely supporting shock wave therapy or eccentric exercise. Characterizing these treatments have demonstrated both generate perturbations within tendon at a frequency of approximately 8-12 Hz. Consequently, it is hypothesized that loading in this frequency range initiates increased anabolic tenocyte behavior, promoting tendon repair. The primary aim of this study was to investigate the effects of 10 Hz perturbations on tenocyte metabolism, comparing gene expression in response to a 10 Hz and 1 Hz loading profile. Tenocytes from healthy and tendinopathic human tendons were seeded into 3D collagen gels and subjected to 15 minutes cyclic strain at 10 Hz or 1 Hz. Tenocytes from healthy tendon showed increased expression of all analyzed genes in response to loading, with significantly increased expression of inflammatory and degradative genes with 10 Hz, relative to 1 Hz loading. By contrast, whilst the response of tenocytes from tendinopathy tendon also increased with 10 Hz loading, the overall response profile was more varied and less intense, possibly indicative of an altered healing response. Through inhibition of the pathway, IL1 was shown to be involved in the degradative and catabolic response of cells to high-frequency loading, abrogating the loading response. This study has demonstrated for the first time that loading at a frequency of 10 Hz may enhance the metabolic response of tenocytes by initiating an immediate degradatory and inflammatory cell response through the IL1 pathway, perhaps as an initial stage of tendon healing.

Distribution of proteins within different compartments of tendon varies according to tendon type.

Although the predominant function of all tendons is to transfer force from muscle to bone and position the limbs, some tendons additionally function as energy stores, reducing the energetic cost of locomotion. To maximise energy storage and return, energy-storing tendons need to be more extensible and elastic than tendons with a purely positional function. These properties are conferred in part by a specialisation of a specific compartment of the tendon, the interfascicular matrix, which enables sliding and recoil between adjacent fascicles. However, the composition of the interfascicular matrix is poorly characterised and we therefore tested the hypothesis that the distribution of elastin and proteoglycans differs between energy-storing and positional tendons, and that protein distribution varies between the fascicular matrix and the interfascicular matrix, with localisation of elastin and lubricin to the interfascicular matrix. Protein distribution in the energy-storing equine superficial digital flexor tendon and positional common digital extensor tendon was assessed using histology and immunohistochemistry. The results support the hypothesis, demonstrating enrichment of lubricin in the interfascicular matrix in both tendon types, where it is likely to facilitate interfascicular sliding. Elastin was also localised to the interfascicular matrix, specifically in the energy-storing superficial digital flexor tendon, which may account for the greater elasticity of the interfascicular matrix in this tendon. A differential distribution of proteoglycans was identified between tendon types and regions, which may indicate a distinct role for each of these proteins in tendon. These data provide important advances into fully characterising structure-function relationships within tendon.

Cyclical strain modulates metalloprotease and matrix gene expression in human tenocytes via activation of TGFß.

Tendinopathies are a range of diseases characterised by degeneration and chronic tendon pain and represent a significant cause of morbidity. Relatively little is known about the underlying mechanisms; however onset is often associated with physical activity. A number of molecular changes have been documented in tendinopathy such as a decrease in overall collagen content, increased extracellular matrix turnover and protease activity. Metalloproteinases are involved in the homeostasis of the extracellular matrix and expression is regulated by mechanical strain. The aims of this study were to determine the effects of strain upon matrix turnover by measuring metalloproteinase and matrix gene expression and to elucidate the mechanism of action. Primary Human Achilles tenocytes were seeded in type I rat tail collagen gels in a Flexcell™ tissue train system and subjected to 5% cyclic uniaxial strain at 1Hz for 48h. TGFß1 and TGFßRI inhibitor were added to selected cultures. RNA was measured using qRT-PCR and TGFß protein levels were determined using a cell based luciferase assay. We observed that mechanical strain regulated the mRNA levels of multiple protease and matrix genes anabolically, and this regulation mirrored that seen with TGFß stimulation alone. We have also demonstrated that the inhibition of the TGFß signalling pathway abrogated the strain induced changes in mRNA and that TGFß activation, rather than gene expression, was increased with mechanical strain. We concluded that TGFß activation plays an important role in mechanotransduction. Targeting this pathway may have its place in the treatment of tendinopathy.

MMP-14 and MMP-2 are key metalloproteases in Dupuytren's disease fibroblast-mediated contraction.

Dupuytren's disease (DD) is a common fibrotic condition of the palmar fascia, leading to deposition of collagen-rich cords and progressive flexion of the fingers. The molecular mechanisms underlying the disease are poorly understood. We have previously shown altered expression of extracellular matrix-degrading proteases (matrix metalloproteases, MMPs, and 'a disintegrin and metalloprotease domain with thrombospondin motifs', ADAMTS, proteases) in palmar fascia from DD patients compared to control and shown that the expression of a sub-set of these genes correlates with post-operative outcome. In the current study we used an in vitro model of collagen contraction to identify the specific proteases which mediate this effect. We measured the expression of all MMPs, ADAMTSs and their inhibitors in fibroblasts derived from the palmar fascia of DD patients, both in monolayer culture and in the fibroblast-populated collagen lattice (FPCL) model of cell-mediated contraction. Key proteases, previously identified in our tissue studies, were expressed in vitro and regulated by tension in the FPCL, including MMP1, 2, 3, 13 and 14. Knockdown of MMP2 and MMP14 (but not MMP1, 3 and 13) inhibited cell-mediated contraction, and knockdown of MMP14 inhibited proMMP-2 activation. Interestingly, whilst collagen is degraded during the FPCL assay, this is not altered upon knockdown of any of the proteases examined. We conclude that MMP-14 (via its ability to activate proMMP-2) and MMP-2 are key proteases in collagen contraction mediated by fibroblasts in DD patients. These proteases may be drug targets or act as biomarkers for disease progression.

Changes in matrix protein biochemistry and the expression of mRNA encoding matrix proteins and metalloproteinases in posterior tibialis tendinopathy.

OBJECTIVES: Adult-acquired flat foot secondary to a dysfunctional posterior tibialis tendon (PTT) is often treated by surgical transfer of the flexor digitorum longus tendon (FDLT). In this study, the authors compared normal PTT, stage II dysfunctional PTT and replacement FDLT, aiming to define changes in collagen modification, glycosaminoglycan (GAG) and the expression of matrix and metalloproteinase mRNA. METHODS: Normal PTTs were obtained from patients with no history of tendon problems. Samples of dysfunctional PTT and replacement FDLT tissue were obtained from patients undergoing surgical reconstruction. Tissue samples were analysed for total collagen and GAG, pentosidine and collagen cross-links. Total RNA was assayed for mRNA encoding matrix proteins and metalloproteinases, using real-time reverse transcription PCR. Differences between clinical groups were assessed using non-parametric statistics. RESULTS: Dysfunctional PTT contained higher levels of GAG and lower levels of pentosidine than normal PTT or FDLT. In contrast, collagen in FDLT contained fewer ketoimine and more aldimine cross-links than either normal or dysfunctional PTT. mRNA encoding types I and III collagens, aggrecan, biglycan, matrix metalloproteinase (MMP)-2, -13 and -23, and a disintegrin and metalloproteinase (ADAM)-12L each showed increased levels in dysfunctional PTT compared with either normal PTT or (except MMP-13) FDLT. In contrast, MMP-3 and ADAM with thrombospondin domain (ADAMTS)-5 mRNA were lower in both dysfunctional PTT and FDLT than in normal PTT, while ADAMTS-1 mRNA was lower in dysfunctional PTT than in FDLT. CONCLUSIONS: Stage II dysfunctional PTT shows biochemical and molecular changes consistent with a chronic remodelling of the extracellular matrix, rather than rupture, while the replacement FDLT resembles normal PTT in many, but not all, parameters.

Increased expression of IL-6 family members in tendon pathology.

OBJECTIVES: Histological examination of pathological tendon generally does not reveal signs of inflammation. However, the inflammatory cytokine IL-6 has been shown to be expressed in ruptured rotator cuff tendon. The aim of this study was to investigate the expression of IL-6 family members in painful posterior tibialis tendon (PTT) and in painful and ruptured Achilles tendon (AT) compared with normal tendon. METHODS: AT samples were obtained from cadavers (normal) or from patients undergoing surgical procedures to treat chronic painful tendinopathy or ruptured tendon. PTT samples were obtained from patients undergoing surgery for other reasons (normal) and from patients with PTT dysfunction (painful). Total RNA was extracted and mRNA expression was analysed by quantitative real-time PCR. RESULTS: Collagen type I α-chain I (COL1A1) expression was increased in both painful PTT and AT compared with normal. Ciliary neurotrophic factor levels were increased in painful PTT only. In the painful AT, cyclooxygenase-2 (COX2) and IL-6 expression increased compared with normal. In the ruptured AT, levels of VEGF A, COX2, oncostatin-M, leukaemia inhibitory factor and IL-6 expression were higher compared with both normal and painful AT. IL-6R expression decreased in both painful and ruptured AT compared with normal. CONCLUSION: Painful AT and PTT show different expression patterns, indicating a substantial difference between those two tendinopathies. Inflammatory markers are up-regulated in painful and particularly in ruptured AT, pointing towards a role of inflammation not only in rupture healing, but also in Achilles tendinopathy.

Induction of Tendon-Specific Markers in Adipose-Derived Stem Cells in Serum-Free Culture Conditions.

IMPACT STATEMENT: Herein, we describe the tenogenic effect of bone morphogenetic protein-12 and transforming growth factor-ß1 in cultured adipose-derived stem cells (ADSCs) in serum-free conditions. This culture system provides an insight into serum-free culture conditions in stem cell differentiation protocols. A positive response of the ADSCs to the tenogenic induction was observed. In particular, the different growth factors used in this study displayed notable differences both on the gene and on the protein expression of the tendon-specific markers. The results underline the positive outcome of the serum removal in tenogenic differentiation protocols, contributing to the development of future cell-based therapies for tendon regeneration and repair.

Stiffness post-total knee replacement: A proof of principle study investigating the effect of gene expression analysis of markers of fibrosis.

BACKGROUND: To establish proof of principle of a link between phenotypic expression and stiffness after TKR. METHODS: From 100 patients, genetic expression of markers of fibrosis was performed for 15 synovial samples from patients categorised as 'best post-operative range of movement (ROM)' and 15 samples from patients with 'worst ROM'. These markers included Matrix Metalloproteinases (MMPs), A Disintegrin and Metalloproteinases with Thrombospondin (ADAMTS) and Tissue Inhibitors of Matrix Metalloproteinases (TIMPs). Genetic marker data were compared to Oxford Knee Scores (OKS) and Pain Catastrophizing Scores (PCS). RESULTS: Quantitative markers for gene expression demonstrated more outliers in stiff compared to non-stiff knees, suggesting a greater imbalance in pro- and anti-fibrotic markers in stiff knees. Whilst there was a significant difference in the range of post-operative knee flexion (p = 0.001) and extension (p = 0.001), there was no statistically significant difference between stiff and non-stiff knees in pre-operative or post-operative OKS (p ≥ 0.06). There was no difference in the individual components of the individual PCS score items nor the PCS total scores when stiff and non-stiff knees were compared (p > 0.05). CONCLUSION: Biological factors, namely gene expression of MMPs, TIMPs and ADAMTS, may contribute towards post-TKR stiffness. This now warrants further investigation to better understand this relationship based on larger, multi-centre, cohorts. LEVEL OF EVIDENCE: Level 3.

The role of proteases in pathologies of the synovial joint.

Synovial (diarthrodial) joints are employed within the body to provide skeletal mobility and have a characteristic structure adapted to provide a smooth almost frictionless surface for articulation. Pathologies of the synovial joint are an important cause of patient morbidity and can affect each of the constituent tissues. A common feature of these pathologies is degenerative changes in the structure of the tissue which is mediated, at least in part, by proteolytic activity. Most tissues of the synovial joint are composed primarily of extracellular matrix and key pathological roles in the degeneration of this matrix are performed by metalloproteinases such as matrix metallproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS). However, other proteases such as cathepsin K are likely to play an important role, especially in bone turnover. In addition to the cleavage of structural proteins, proteolytic activities are employed to regulate the activity of other proteases, growth factors, cytokines and other inflammatory mediators. Proteases combine to form complex regulatory networks, the correct functioning of which is required for tissue homeostasis and the imbalance of which may be a feature of pathology. A precise understanding of the proteases involved in these networks is required for a true understanding of the associated pathology.

The regulation of aggrecanase ADAMTS-4 expression in human Achilles tendon and tendon-derived cells.

Several members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family have been identified as aggrecanases, whose substrates include versican, the principal large proteoglycan in the tendon extracellular matrix. We have characterized the expression of ADAMTS-4 in human Achilles tendon and tendon-derived cells. ADAMTS-4 mRNA levels were higher in ruptured tendon compared with normal tendon or chronic painful tendinopathy. In tissue extracts probed by Western blotting, mature ADAMTS-4 (68 kDa) was detected only in ruptured tendons, while processed ADAMTS-4 (53 kDa) was detected also in chronic painful tendinopathy and in normal tendon. In cultured Achilles tendon cells, transforming growth factor-beta (TGF-beta) stimulated ADAMTS-4 mRNA expression (typically 20-fold after 24 h), while interleukin-1 induced a smaller, shorter-term stimulation which synergised markedly with that induced by TGF-beta. Increased levels of immunoreactive proteins consistent with mature and processed forms of ADAMTS-4 were detected in TGF-beta-stimulated cells. ADAMTS-4 mRNA was expressed at higher levels by tendon cells in collagen gels than in monolayer cultures. In contrast, the expression of ADAMTS-1 and -5 mRNA was lower in collagen gels compared with monolayers, and these mRNA showed smaller or opposite responses to growth factors and cytokines compared with that of ADAMTS-4 mRNA. We conclude that both ADAMTS-4 mRNA and ADAMTS-4 protein processing may be differentially regulated in normal and damaged tendons and that both the matrix environment and growth factors such as TGF-beta are potentially important factors controlling ADAMTS aggrecanase activities in tendon pathology.

Tendinopathy--from basic science to treatment.

Chronic tendon pathology (tendinopathy), although common, is difficult to treat. Tendons possess a highly organized fibrillar matrix, consisting of type I collagen and various 'minor' collagens, proteoglycans and glycoproteins. The tendon matrix is maintained by the resident tenocytes, and there is evidence of a continuous process of matrix remodeling, although the rate of turnover varies at different sites. A change in remodeling activity is associated with the onset of tendinopathy. Major molecular changes include increased expression of type III collagen, fibronectin, tenascin C, aggrecan and biglycan. These changes are consistent with repair, but they might also be an adaptive response to changes in mechanical loading. Repeated minor strain is thought to be the major precipitating factor in tendinopathy, although further work is required to determine whether it is mechanical overstimulation or understimulation that leads to the change in tenocyte activity. Metalloproteinase enzymes have an important role in the tendon matrix, being responsible for the degradation of collagen and proteoglycan in both healthy patients and those with disease. Metalloproteinases that show increased expression in painful tendinopathy include ADAM (a disintegrin and metalloproteinase)-12 and MMP (matrix metalloproteinase)-23. The role of these enzymes in tendon pathology is unknown, and further work is required to identify novel and specific molecular targets for therapy.

A Clinical, Biological, and Biomaterials Perspective into Tendon Injuries and Regeneration.

Tendon injury is common and debilitating, and it is associated with long-term pain and ineffective healing. It is estimated to afflict 25% of the adult population and is often a career-ending disease in athletes and racehorses. Tendon injury is associated with high morbidity, pain, and long-term suffering for the patient. Due to the low cellularity and vascularity of tendon tissue, once damage has occurred, the repair process is slow and inefficient, resulting in mechanically, structurally, and functionally inferior tissue. Current treatment options focus on pain management, often being palliative and temporary and ending in reduced function. Most treatments available do not address the underlying cause of the disease and, as such, are often ineffective with variable results. The need for an advanced therapeutic that addresses the underlying pathology is evident. Tissue engineering and regenerative medicine is an emerging field that is aimed at stimulating the body's own repair system to produce de novo tissue through the use of factors such as cells, proteins, and genes that are delivered by a biomaterial scaffold. Successful tissue engineering strategies for tendon regeneration should be built on a foundation of understanding of the molecular and cellular composition of healthy compared with damaged tendon, and the inherent differences seen in the tissue after disease. This article presents a comprehensive clinical, biological, and biomaterials insight into tendon tissue engineering and regeneration toward more advanced therapeutics.

Fascicles and the interfascicular matrix show decreased fatigue life with ageing in energy storing tendons.

Tendon is composed of rope-like fascicles bound together by interfascicular matrix (IFM). The IFM is critical for the function of energy storing tendons, facilitating sliding between fascicles to allow these tendons to cyclically stretch and recoil. This capacity is required to a lesser degree in positional tendons. We have previously demonstrated that both fascicles and IFM in energy storing tendons have superior fatigue resistance compared with positional tendons, but the effect of ageing on the fatigue properties of these different tendon subunits has not been determined. Energy storing tendons become more injury-prone with ageing, indicating reduced fatigue resistance, hence we tested the hypothesis that the decline in fatigue life with ageing in energy storing tendons would be more pronounced in the IFM than in fascicles. We further hypothesised that tendon subunit fatigue resistance would not alter with ageing in positional tendons. Fascicles and IFM from young and old energy storing and positional tendons were subjected to cyclic fatigue testing until failure, and mechanical properties were calculated. The results show that both IFM and fascicles from the SDFT exhibit a similar magnitude of reduced fatigue life with ageing. By contrast, the fatigue life of positional tendon subunits was unaffected by ageing. The age-related decline in fatigue life of tendon subunits in energy storing tendons is likely to contribute to the increased risk of injury in aged tendons. Full understanding of the mechanisms resulting in this reduced fatigue life will aid in the development of treatments and interventions to prevent age-related tendinopathy. STATEMENT OF SIGNIFICANCE: Understanding the effect of ageing on tendon-structure function relationships is crucial for the development of effective preventative measures and treatments for age-related tendon injury. In this study, we demonstrate for the first time that the fatigue resistance of the interfascicular matrix decreases with ageing in energy storing tendons. This is likely to contribute to the increased risk of injury in aged tendons. Full understanding of the mechanisms that result in this reduced fatigue resistance will aid in the development of treatments and interventions to prevent age-related tendinopathy.

SB-399885 is a potent, selective 5-HT6 receptor antagonist with cognitive enhancing properties in aged rat water maze and novel object recognition models.

SB-399885 (N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)benzenesulfonamide) has high affinity for human recombinant and native 5-HT(6) receptors, with pK(i) values 9.11+/-0.03 and 9.02+/-0.05, respectively and is a potent competitive antagonist (pA(2) 7.85+/-0.04). It displays over 200-fold selectivity for the 5-HT(6) receptor over all other receptors, ion channels and enzymes tested to date. SB-399885 inhibited ex vivo [(125)I]SB-258585 (4-Iodo-N-[4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-benzenesulfonamide) binding with an ED(50) of 2.0+/-0.24 mg/kg p.o. in rats. It had a minimum effective dose of 1 mg/kg p.o. in a rat maximal electroshock seizure threshold test and a long duration of action, overall demonstrating an excellent pharmacokinetic-pharmacodynamic correlation. Repeated administration of this agent (10 mg/kg p.o., b.i.d. for 7 days) significantly reversed a scopolamine-induced deficit (0.5 mg/kg i.p.) in a rat novel object recognition paradigm. Moreover, in aged rats (22 months old) SB-399885 (10 mg/kg p.o., b.i.d. for 7 days) fully reversed the age-dependent deficit in water maze spatial learning compared to vehicle-treated age-matched controls and significantly improved recall of the task measured by increases in the searching of the target quadrant on post-training days 1, 3 and 7. In vivo microdialysis in the rat medial prefrontal cortex demonstrated that acute SB-399885 (10 mg/kg p.o.) significantly increased extracellular acetylcholine levels. These data demonstrate that SB-399885 is a potent, selective, brain penetrant, orally active 5-HT(6) receptor antagonist with cognitive enhancing properties that are likely to be mediated by enhancements of cholinergic function. These studies provide further support for the potential therapeutic utility of 5-HT(6) receptor antagonists in disorders characterised by cognitive deficits such as Alzheimer's disease and schizophrenia.

Fascicles and the interfascicular matrix show adaptation for fatigue resistance in energy storing tendons.

UNLABELLED: Tendon is composed of rope-like fascicles, bound together by interfascicular matrix (IFM). Our previous work shows that the IFM is critical for tendon function, facilitating sliding between fascicles to allow tendons to stretch. This function is particularly important in energy storing tendons, which experience extremely high strains during exercise, and therefore require the capacity for considerable inter-fascicular sliding and recoil. This capacity is not required in positional tendons. Whilst we have previously described the quasi-static properties of the IFM, the fatigue resistance of the IFM in functionally distinct tendons remains unknown. We therefore tested the hypothesis that fascicles and IFM in the energy storing equine superficial digital flexor tendon (SDFT) are more fatigue resistant than those in the positional common digital extensor tendon (CDET). Fascicles and IFM from both tendon types were subjected to cyclic fatigue testing until failure, and mechanical properties were calculated. The results demonstrated that both fascicles and IFM from the energy storing SDFT were able to resist a greater number of cycles before failure than those from the positional CDET. Further, SDFT fascicles and IFM exhibited less hysteresis over the course of testing than their counterparts in the CDET. This is the first study to assess the fatigue resistance of the IFM, demonstrating that IFM has a functional role within tendon and contributes significantly to tendon mechanical properties. These data provide important advances into fully characterising tendon structure-function relationships. STATEMENT OF SIGNIFICANCE: Understanding tendon-structure function relationships is crucial for the development of effective preventative measures and treatments for tendon injury. In this study, we demonstrate for the first time that the interfascicular matrix is able to withstand a high degree of cyclic loading, and is specialised for improved fatigue resistance in energy storing tendons. These findings highlight the importance of the interfascicular matrix in the function of energy storing tendons, and potentially provide new avenues for the development of treatments for tendon injury which specifically target the interfascicular matrix.

Chronic tendon pathology: molecular basis and therapeutic implications.

Tendons are frequently affected by chronic pain or rupture. Many causative factors have been implicated in the pathology, which until relatively recently was under-researched and poorly understood. There is now a greater knowledge of the molecular basis of tendon disease. Most tendon pathology (tendinopathy) is associated with degeneration, which is thought to be an active, cell-mediated process involving increased turnover and remodelling of the tendon extracellular matrix. Degradation of the tendon matrix is mediated by a variety of metalloproteinase enzymes, including matrix metalloproteinases and 'aggrecanases'. Neuropeptides and other factors released by stimulated cells or nerve endings in or around the tendon might influence matrix turnover, and could provide novel targets for therapeutic intervention.

Nonpeptidic urotensin-II receptor antagonists I: in vitro pharmacological characterization of SB-706375.

1. SB-706375 potently inhibited [(125)I]hU-II binding to both mammalian recombinant and 'native' UT receptors (K(i) 4.7+/-1.5 to 20.7+/-3.6 nM at rodent, feline and primate recombinant UT receptors and K(i) 5.4+/-0.4 nM at the endogenous UT receptor in SJRH30 cells). 2. Prior exposure to SB-706375 (1 microM, 30 min) did not alter [(125)I]hU-II binding affinity or density in recombinant cells (K(D) 3.1+/-0.4 vs 5.8+/-0.9 nM and B(max) 3.1+/-1.0 vs 2.8+/-0.8 pmol mg(-1)) consistent with a reversible mode of action. 3. The novel, nonpeptidic radioligand [(3)H]SB-657510, a close analogue of SB-706375, bound to the monkey UT receptor (K(D) 2.6+/-0.4 nM, B(max) 0.86+/-0.12 pmol mg(-1)) in a manner that was inhibited by both U-II isopeptides and SB-706375 (K(i) 4.6+/-1.4 to 17.6+/-5.4 nM) consistent with the sulphonamides and native U-II ligands sharing a common UT receptor binding domain. 4. SB-706375 was a potent, competitive hU-II antagonist across species with pK(b) 7.29-8.00 in HEK293-UT receptor cells (inhibition of [Ca(2+)](i)-mobilization) and pK(b) 7.47 in rat isolated aorta (inhibition of contraction). SB-706375 also reversed tone established in the rat aorta by prior exposure to hU-II (K(app) approximately 20 nM). 5. SB-706375 was a selective U-II antagonist with >/=100-fold selectivity for the human UT receptor compared to 86 distinct receptors, ion channels, enzymes, transporters and nuclear hormones (K(i)/IC(50)>1 microM). Accordingly, the contractile responses induced in isolated aortae by KCl, phenylephrine, angiotensin II and endothelin-1 were unaltered by SB-706375 (1 microM). 6. In summary, SB-706375 is a high-affinity, surmountable, reversible and selective nonpeptide UT receptor antagonist with cross-species activity that will assist in delineating the pathophysiological actions of U-II in mammals.