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Armillaria and Desarmillaria spp. are causal agents of a devastating root-borne disease of peach. Breeding resistant rootstock requires a reliable screening tool. An in vitro co-culture screen designed for almond was modified by replacing agar-gelled medium with a more aerated phenolic foam and combining resistant and susceptible rootstocks (i.e., common garden experiment) and minimizes variation in inoculum pressure or rooting substrate among replicate vessels. Eight Prunus rootstocks tested (peach, plum, peach × plum, and choke cherry) were rooted and had no decline in health. Susceptible peach rootstock, ‘GF 305’, was cultured for 15 wk in phenolic foam in the same vessel with a resistant peach × plum hybrid, ‘MP-29’, inoculated with Armillaria mellea at week 5, that led to more severe shoot symptoms in the former after an additional 8 wk. This method accommodated peach genotypes that were difficult to root in agar medium. The difference during a uniform challenge with the A. mellea fungus recapitulates resistant/susceptible reactions. The phenolic foam-based co-culture method will work on many Prunus spp. of potential use in rootstock breeding.

Peptide therapeutics are increasingly used in the treatment of disease, but their administration by injection reduces patient compliance and convenience, especially for chronic diseases. Thus, oral administration of a peptide therapeutic represents a significant advance in medicine, but is challenged by gastrointestinal instability and ineffective uptake into the circulation. Here, we have used glucagon-like peptide-1 (GLP-1) as a model peptide therapeutic for treating obesity-linked type 2 diabetes, a common chronic disease. We describe a comprehensive multidisciplinary approach leading to the development of MEDI7219, a GLP-1 receptor agonist (GLP-1RA) specifically engineered for oral delivery. Sites of protease/peptidase vulnerabilities in GLP-1 were removed by amino acid substitution and the peptide backbone was bis-lipidated to promote MEDI7219 reversible plasma protein binding without affecting potency. A combination of sodium chenodeoxycholate and propyl gallate was used to enhance bioavailability of MEDI7219 at the site of maximal gastrointestinal absorption, targeted by enteric-coated tablets. This synergistic approach resulted in MEDI7219 bioavailability of ~ 6% in dogs receiving oral tablets. In a dog model of obesity and insulin resistance, MEDI7219 oral tablets significantly decreased food intake, body weight and glucose excursions, validating the approach. This novel approach to the development of MEDI7219 provides a template for the development of other oral peptide therapeutics.

Nutrient excess, a major driver of obesity, diminishes hypothalamic responses to exogenously administered leptin, a critical hormone of energy balance. Here, we aimed to identify a physiological signal that arises from excess caloric intake and negatively controls hypothalamic leptin action. We found that deficiency of the gastric inhibitory polypeptide receptor (Gipr) for the gut-derived incretin hormone GIP protected against diet-induced neural leptin resistance. Furthermore, a centrally administered antibody that neutralizes GIPR had remarkable antiobesity effects in diet-induced obese mice, including reduced body weight and adiposity, and a decreased hypothalamic level of SOCS3, an inhibitor of leptin actions. In contrast, centrally administered GIP diminished hypothalamic sensitivity to leptin and increased hypothalamic levels of Socs3. Finally, we show that GIP increased the active form of the small GTPase Rap1 in the brain and that its activation was required for the central actions of GIP. Altogether, our results identify GIPR/Rap1 signaling in the brain as a molecular pathway linking overnutrition to the control of neural leptin actions.

Petunia × hybrida Vilm. is a fast-growing ornamental plant that was cultured under varying storage conditions to address seasonal fluctuations in microcutting demand. The effects of storage period (16 to 32 wk), temperature (15 to 23°C), low and high light intensity, sucrose (1 to 5% w/v), and mannitol (0 to 4% w/v) in factorial arrangements were analyzed. Stored and non-stored shoots were compared for microcutting production, harvested twice at 3-wk intervals, and were subsequently transferred to the greenhouse for 17 d. Nearly all plants from 16-wk storage survived well at all treatment conditions and the quality and quantity of microcuttings were enhanced from shoots stored at 15°C and 3% (w/v) sucrose, without mannitol for all storage periods. Another experiment tested 11- to 25-wk storage period with Petunia hybrida ‘Ragtime’ and ‘Suncatcher’ at 12°C and low light intensity. Repeated cycles of microcutting at 2-wk intervals were extended with ex vitro rooting in the greenhouse for 15 d. More and better quality microcuttings were obtained from the second and third cutting cycles than from the first or fourth cycles. By reducing temperature and light intensity, Petunia hybrida was successfully stored for 32 wk (without mannitol). A seasonal schedule with a short production window, followed by cutting large numbers of high-quality shoot-tips, could be affected and efficiently managed through storage. The value of stored shoots was enhanced by extending the number of times a shoot could be cut over repeated cutting cycles with a gain in microcutting quality.

A disposable, flexible film vessel was demonstrated for liquid culture micrporopagation of Poacea × ‘Cape Breeze’ and a micropropagation process was optimized for this system. Process factors involving media volume and plant density; mineral factors of ammonium and phosphate concentrations; and the important organic chemicals sucrose and benzyladenine; were simultaneously varied in the presence and absence of an inert paper substrate (40 treatment combinations for the six continuous factors, duplicated for the nominal factor, paper). Plant multiplication was enhanced by having fewest plants per vessel (10) with the largest volume of medium (90 mL), with the greatest concentrations of benyzladenine (10 µM) and phosphate (6.25 mM), in the absence of the paper. Optimal ex vitro growth, measured by dry mass increase on the mist bed required plants that had been grown under very different in vitro conditions (the smallest volume of medium per vessel (30 mL), fewest plants per vessel (10), low benzyladenine (1.6 µM), lowest phosphate (1.25 mM), in the presence of paper). Media that produced the largest in vitro plants produced plants that rapidly increased in dry mass in the greenhouse. Increasing the number of plants per vessel (30) increases the economy of the system with greater numbers of plants, but they are smaller and grow more slowly than the low-density cultures. In a small experiment using 80 vessels, separate optimal conditions in both process and media formulation were determined for both stage II and stage III cultures of Poacea. The analysis identified most important factors that drive the responses, from other significant responses with less impact. A liquid culture micropropagation system for Poacea in flexible-film vessels was optimized for process and nutritional factors in both stages II and III in a small, response surface method experiment.

Non-alcoholic fatty liver disease and steatohepatitis are highly associated with obesity and type 2 diabetes mellitus. Cotadutide, a glucagon-like protein-1 receptor (GLP-1R) and glucagon receptor (GCGR) agonist, was shown to reduce blood glycaemia, body weight and hepatic steatosis in people with type 2 diabetes mellitus. Here, we demonstrate that the effects of cotadutide in reducing body weight and food intake and improving glucose control are predominantly mediated through Glp-1 signalling, whereas its action on the liver to reduce lipid content, drive glycogen flux and improve mitochondrial turnover and function are directly mediated through Gcg signalling. This was confirmed by the identification of phosphorylation sites on key lipogenic and glucose metabolism enzymes in liver of mice treated with cotadutide. Complementary metabolomic and transcriptomic analyses implicated lipogenic, fibrotic and inflammatory pathways, consistent with a unique therapeutic contribution of GCGR agonism by cotadutide in vivo. Notably, cotadutide also alleviated fibrosis to a greater extent than liraglutide or obeticholic acid, despite dose adjustment to achieve similar weight loss in two preclinical mouse models of NASH. Thus, cotadutide, via direct hepatic (GcgR) and extrahepatic (Glp-1R) effects, exerts multifactorial improvement in liver function and is a promising therapeutic option for the treatment of steatohepatitis.

Objective Glucose-dependent insulinotropic polypeptide is an intestinally derived hormone that is essential for normal metabolic regulation. Loss of the GIP receptor (GIPR) through genetic elimination or pharmacological antagonism reduces body weight and adiposity in the context of nutrient excess. Interrupting GIPR signaling also enhances the sensitivity of the receptor for the other incretin peptide, glucagon-like peptide 1 (GLP-1). The role of GLP-1 compensation in loss of GIPR signaling to protect against obesity has not been directly tested. Methods We blocked the GIPR and GLP-1R with specific antibodies, alone and in combination, in healthy and diet-induced obese (DIO) mice. The primary outcome measure of these interventions was the effect on body weight and composition. Results Antagonism of either the GIPR or GLP-1R system reduced food intake and weight gain during high-fat feeding and enhanced sensitivity to the alternative incretin signaling system. Combined antagonism of both GIPR and GLP-1R produced additive effects to mitigate DIO. Acute pharmacological studies using GIPR and GLP-1R agonists demonstrated both peptides reduced food intake, which was prevented by co-administration of the respective antagonists. Conclusions Disruption of either axis of the incretin system protects against diet-induced obesity in mice. However, combined antagonism of both GIPR and GLP-1R produced additional protection against diet-induced obesity, suggesting additional factors beyond compensation by the complementary incretin axis. While antagonizing the GLP-1 system decreases weight gain, GLP-1R agonists are used clinically to target obesity. Hence, the phenotype arising from loss of function of GLP-1R does not implicate GLP-1 as an obesogenic hormone. By extension, caution is warranted in labeling GIP as an obesogenic hormone based on loss-of-function studies., Highlights • Acute administration of either GIP or GLP-1 reduces food intake inmice, which is blocked by antagonizing antibodies. • Chronic antagonism of the GIPR limits weight gain, improves glucose tolerance, and enhances sensitivity to GLP-1R agonists. • Chronic antagonism of the GLP-1R reduces weight gain and enhances sensitivity to GIPR agonists. • Chronic antagonism of both GIPR and GLP-1R provides additive protections against weight gain when mice are fed a HFD. • Incretin receptor antagonism reduces food intake but does not change energy expenditure.

Gut hormones have long been understood to regulate food intake and metabolism. Bariatric surgery significantly elevates circulating gut hormone levels and is proven to affect acute remission of type 2 diabetes before any weight loss is observed. Subsequent weight loss is accrued over weeks to months but is sustained into the long term. Hence, there exists great enthusiasm to recapitulate these changes in gut hormones in the form of novel combination drugs for type 2 diabetes and obesity.

The use of peptides as therapeutic agents is undergoing a renaissance with the expectation of new drugs with enhanced levels of efficacy and safety. Their clinical potential will be only fully realised once their physicochemical and pharmacokinetic properties have been precisely controlled. Here we demonstrate a reversible peptide self-assembly strategy to control and prolong the bioactivity of a native peptide hormone in vivo. We show that oxyntomodulin, a peptide with potential to treat obesity and diabetes, self-assembles into a stable nanofibril formulation which subsequently dissociates to release active peptide and produces a pharmacological effect in vivo. The subcutaneous administration of the nanofibrils in rats results in greatly prolonged exposure, with a constant oxyntomodulin bioactivity detectable in serum for at least 5 days as compared to free oxyntomodulin which is undetectable after only 4 h. Such an approach is simple, cost-efficient and generic in addressing the limitations of peptide therapeutics., The clinical potential of peptide therapeutic agents can only be fully realised once their physicochemical and pharmacokinetic properties are precisely controlled. Here the authors show a reversible peptide self-assembly strategy to control and prolong the bioactivity of a native peptide hormone in vivo.

Shoot cultures of Echinacea × Sombrero® “Salsa Red” were rooted in vitro in rigid and flexible film vented vessels, in agar-gelled medium, and in phenolic foam plugs (Oasis® InVitro Express [IVE]). Rooted plants were acclimatized to greenhouse conditions in a peat-based, soilless medium or in a larger cellular plug (Oasis® Rootcubes® Wedge®). The size of the plants leaving the laboratory, the speed of planting in greenhouse medium, and plant growth in greenhouse medium were influenced by both the laboratory and greenhouse media in which they were grown. The largest plants leaving the laboratory were from agar medium. The speed of deflasking and medium removal was greatly improved with IVE replacing agar in laboratory vessels. Planting in the Oasis® Rootcubes® Wedge® allowed a further reduction in planting time compared to planting in peat-based medium. In the greenhouse, plants in IVE/Oasis® Rootcubes® Wedge® grew more quickly and had more leaves of better quality than those from agar, planted in peat-based medium. However, the best plants (based on number of leaves, leaf length, and plug quality) in the greenhouse were from IVE, planted in peat-based medium, a treatment that still required less planting time than agar-based medium. Pre-hardening of in vitro-rooted plantlets using vessel systems that allow for ventilation and easily transferred root systems showed superior greenhouse growth simultaneous with improvements in the efficiency of workers that handled the plants. This is the direct impact of vessel systems and matrix purposefully designed to facilitate this stage of growth.

Bariatric surgery can result in profound weight loss and remission of type 2 diabetes. Following meal consumption post-surgery, patients exhibit an exaggerated increase in the circulating levels of the enteroendocrine L-cell peptides peptide YY (PYY) and glucagon-like peptide-1 (GLP-1). We have developed a long-acting PYY Y2 and GLP-1 receptor agonist (GLP-1-Fc-PYY), which exerted potent effects on improving glucose metabolism and body weight loss in diabetic db/db mice and diet-induced obese (DIO) mice. Administration of GLP-1-Fc-PYY to diabetic db/db mice for 28 days normalized HbA1c levels (4.3%) compared to vehicle controls (9.1%), and exceeded that observed with Fc-GLP-1 monotherapy treatment (7.2%). GLP-1-Fc-PYY administration also preserved circulating insulin levels and increased pancreatic insulin content in db/db mice, which was not observed in mice treated with Fc-GLP-1. Administration of GLP-1-Fc-PYY for 21 days in DIO mice resulted in a 28% (p Disclosure S. Oldham: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. V.G. Howard: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. S. Will: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. D.C. Hornigold: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. C.J. Rhodes: Employee; Self; AstraZeneca, MedImmune. S. Madalli: None. I. Sermadiras: None. J. Naylor: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca. C. Rivera: None.

Type 2 diabetes (T2D) is a complex and progressive disease requiring polypharmacy to manage hyperglycaemia and cardiovascular risk factors. However, most patients do not achieve combined treatment goals. To address this therapeutic gap, we have developed MEDI4166, a novel glucagon-like peptide-1 (GLP-1) receptor agonist peptide fused to a proprotein convertase subtilisin/kexin type 9 (PCSK9) neutralising antibody that allows for glycaemic control and low-density lipoprotein cholesterol (LDL-C) lowering in a single molecule. The fusion has been engineered to deliver sustained peptide activity in vivo in combination with reduced potency, to manage GLP-1 driven adverse effects at high dose, and a favourable manufacturability profile. MEDI4166 showed robust and sustained LDL-C lowering in cynomolgus monkeys and exhibited the anticipated GLP-1 effects in T2D mouse models. We believe MEDI4166 is a novel molecule combining long acting agonist peptide and neutralising antibody activities to deliver a unique pharmacology profile for the management of T2D.

Aims To characterize the pharmacology of MEDI0382, a peptide dual agonist of glucagon-like peptide-1 (GLP-1) and glucagon receptors. Materials and methods MEDI0382 was evaluated in vitro for its ability to stimulate cAMP accumulation in cell lines expressing transfected recombinant or endogenous GLP-1 or glucagon receptors, to potentiate glucose-stimulated insulin secretion (GSIS) in pancreatic β-cell lines and stimulate hepatic glucose output (HGO) by primary hepatocytes. The ability of MEDI0382 to reduce body weight and improve energy balance (i.e. food intake and energy expenditure), as well as control blood glucose, was evaluated in mouse models of obesity and healthy cynomolgus monkeys following single and repeated daily subcutaneous administration for up to 2 months. Results MEDI0382 potently activated rodent, cynomolgus and human GLP-1 and glucagon receptors and exhibited a fivefold bias for activation of GLP-1 receptor versus the glucagon receptor. MEDI0382 produced superior weight loss and comparable glucose lowering to the GLP-1 peptide analogue liraglutide when administered daily at comparable doses in DIO mice. The additional fat mass reduction elicited by MEDI0382 probably results from a glucagon receptor-mediated increase in energy expenditure, whereas food intake suppression results from activation of the GLP-1 receptor. Notably, the significant weight loss elicited by MEDI0382 in DIO mice was recapitulated in cynomolgus monkeys. Conclusions Repeated administration of MEDI0382 elicits profound weight loss in DIO mice and non-human primates, produces robust glucose control and reduces hepatic fat content and fasting insulin and glucose levels. The balance of activities at the GLP-1 and glucagon receptors is considered to be optimal for achieving weight and glucose control in overweight or obese Type 2 diabetic patients.

Routine peptide structure-activity relationship screening requires the serial dilution of peptides to produce full concentration-response curves. Established tip-based protocols involve multiple tip changes and high exposure to plasticware. In the case of peptides, this becomes a challenge, since peptides can adsorb to plastic, resulting in an observed loss of potency. Various methods can be employed to prevent peptide loss during compound handling, such as the inclusion of bovine serum albumin or solvents in assay buffer and the siliconization of plasticware, yet protein binding remains unpredictable. The degree of variation by which peptides will adhere to plasticware can confuse results and cause inaccuracies in potency predictions. We evaluated acoustic noncontact methods for peptide serial dilution and compared it with traditional tip-based methods, on the effect on potency curves for glucagon-like peptide-1 and glucagon peptide analogues. The current study demonstrates the benefits of noncontact dispensing for high-density microplate assay preparation of peptides using nanoliter droplets across our entire drug discovery workflow, from in vitro high-throughput screening to drug exposure determinations from in vivo samples.

Cardiovascular disease is the leading cause of morbidity and mortality in people with type 2 diabetes. MEDI4166 is a proprotein convertase subtilisin/kexin type 9 (PCSK9) antibody and glucagon-like peptide-1 (GLP-1) analogue fusion molecule designed to treat patients with type 2 diabetes who are at risk for cardiovascular disease. In this completed, first-in-human study, we evaluated the safety and efficacy of single or multiple doses of MEDI4166 in participants with type 2 diabetes. In this phase 1 study that was conducted across 11 clinics in the USA, eligible adults had type 2 diabetes, a BMI of ≥25 kg/m2 to ≤42 kg/m2, and LDL-cholesterol levels ≥1.81 mmol/l. Participants were randomised 3:1 to receive MEDI4166 or placebo using an interactive voice/web response system, which blinded all participants, investigators and study site personnel to the study drug administered. In ‘Part A’ of the study, five cohorts of participants received a single s.c. injection of MEDI4166 at 10 mg, 30 mg, 100 mg, 200 mg or 400 mg, or placebo. ‘Part B’ of the study consisted of three cohorts of participants who received an s.c. dose of MEDI4166 once weekly for 5 weeks at 50 mg, 200 mg or 400 mg, or placebo. The primary endpoint in Part A was safety. The co-primary endpoints in Part B were change in LDL-cholesterol levels and area under the plasma glucose concentration–time curve (AUC0–4h) post-mixed-meal tolerance test (MMTT) from baseline to day 36. The pharmacokinetics and immunogenicity of MEDI4166 were also evaluated. MEDI4166 or placebo was administered to n = 30 or n = 10 participants, respectively, in Part A of the study, and n = 48 or n = 15 participants, respectively, in Part B. The incidence of treatment-emergent adverse events (TEAEs) were comparable between MEDI4166 and placebo in both Part A (60% vs 50%) and Part B (79% vs 87%) of the study. Common TEAEs with MEDI4166 included injection-site reactions, diarrhoea and headache; there was no evidence for dose-related increases in TEAEs. In Part B of the study, at all tested doses of MEDI4166, there was a significant decrease in LDL-cholesterol levels vs placebo (least squares mean [95% CI]; MEDI4166 50 mg, −1.25 [−1.66, −0.84]; MEDI4166 200 mg, −1.97 [−2.26, −1.68]; MEDI4166 400 mg, −1.96 [−2.23, −1.70]; placebo, −0.03 [−0.35, 0.28]; all p 0.05). MEDI4166 was associated with a pharmacokinetic profile supportive of weekly dosing and an overall treatment-induced anti-drug antibody-positive rate of 22%. MEDI4166 was associated with an acceptable tolerability profile and significantly decreased LDL-cholesterol levels in a dose-dependent manner in overweight or obese patients with type 2 diabetes. However, there were no significant reductions in postprandial glucose levels at any dose of MEDI4166. ClinicalTrials.gov NCT02524782 This study was funded by MedImmune LLC, Gaithersburg, MD, USA.

AIMS/HYPOTHESIS: Glucagon like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion by binding to GLP-1 receptors (GLP1Rs) on pancreatic beta cells. GLP-1 mimetics are used in the clinic for the treatment of type 2 diabetes, but despite their therapeutic success, several clinical effects of GLP-1 remain unexplained at a mechanistic level, particularly in extrapancreatic tissues. The aim of this study was to generate and characterise a monoclonal antagonistic antibody for the GLP1R for use in vivo. METHODS: A naive phage display selection strategy was used to isolate single-chain variable fragments (ScFvs) that bound to GLP1R. The ScFv with the highest affinity, Glp1R0017, was converted into a human IgG1 and characterised further. In vitro antagonistic activity was assessed in a number of assays: a cAMP-based homogenous time-resolved fluorescence assay in GLP1R-overexpressing cell lines, a live cell cAMP imaging assay and an insulin secretion assay in INS-1 832/3 cells. Glp1R0017 was further tested in immunostaining of mouse pancreas, and the ability of Glp1R0017 to block GLP1R in vivo was assessed by both IPGTT and OGTT in C57/Bl6 mice. RESULTS: Antibodies to GLP1R were selected from naive antibody phage display libraries. The monoclonal antibody Glp1R0017 antagonised mouse, human, rat, cynomolgus monkey and dog GLP1R. This antagonistic activity was specific to GLP1R; no antagonistic activity was found in cells overexpressing the glucose-dependent insulinotropic peptide receptor (GIPR), glucagon like peptide-2 receptor or glucagon receptor. GLP-1-stimulated cAMP and insulin secretion was attenuated in INS-1 832/3 cells by Glp1R0017 incubation. Immunostaining of mouse pancreas tissue with Glp1R0017 showed specific staining in the islets of Langerhans, which was absent in Glp1r knockout tissue. In vivo, Glp1R0017 reversed the glucose-lowering effect of liraglutide during IPGTTs, and reduced glucose tolerance by blocking endogenous GLP-1 action in OGTTs. CONCLUSIONS/INTERPRETATION: Glp1R0017 is a monoclonal antagonistic antibody to the GLP1R that binds to GLP1R on pancreatic beta cells and blocks the actions of GLP-1 in vivo. This antibody holds the potential to be used in investigating the physiological importance of GLP1R signalling in extrapancreatic tissues where cellular targets and signalling pathways activated by GLP-1 are poorly understood.

Factors affecting the micropropagation of Veratrum californicum, a slow-growing species that is a potentially valuable source of cyclopamine, were investigated. Sterile cultures were initiated on modified Murashige and Skoog medium, and clones from individual donor plants were assigned to experimental conditions when approximately 100 shoots of each clone were available. The effects of temperature, light quality, and plant growth regulators on multiplication and survival were assessed. Four clones from which large greenhouse populations were obtained were selected for in-depth analysis. When shoots were cultured at 10°C and 16°C, multiplication ratios consistently >1 were observed from three of four clones and two of four clones, respectively, during the five-subculture cycles. None of the clones stably increased when cultured at 24°C, and plants from this treatment did not survive acclimatization in the greenhouse. Only one clone showed increased multiplication ratios in response to plant growth regulator treatments, with maximum multiplication when shoots were cultured with 9 μM benzyladenine and 0.5 μM naphthaleneacetic acid. Light quality in the laboratory did not affect multiplication ratio but did affect subsequent greenhouse survival. The size of plants derived from culture was most often equivalent (65% of 1,271) to 3-yr-old seed-derived plants. Although the growth of clones during acclimatization differed, plants derived from cultures incubated at 16°C had the best rates of overall greenhouse survival. Temperature and light treatments in vitro critical to long-term plant survival were demonstrated and will assist the establishment of a mass propagation system for V. californicum.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Background and Purpose The Sigma-1 receptor (Sig1R) impacts on calcium ion signalling and has a plethora of ligands. This study investigated Sig1R and its ligands in relation to endogenous calcium events of endothelial cells and transient receptor potential (TRP) channels. Experimental Approach Intracellular calcium and patch clamp measurements were made from human saphenous vein endothelial cells and HEK 293 cells expressing exogenous human TRPC5, TRPM2 or TRPM3. Sig1R ligands were applied and short interfering RNA was used to deplete Sig1R. TRP channels tagged with fluorescent proteins were used for subcellular localization studies. Key Results In endothelial cells, 10–100 μM of the Sig1R antagonist BD1063 inhibited sustained but not transient calcium responses evoked by histamine. The Sig1R agonist 4-IBP and related antagonist BD1047 were also inhibitory. The Sig1R agonist SKF10047 had no effect. Sustained calcium entry evoked by VEGF or hydrogen peroxide was also inhibited by BD1063, BD1047 or 4-IBP, but not SKF10047. 4-IBP, BD1047 and BD1063 inhibited TRPC5 or TRPM3, but not TRPM2. Inhibitory effects of BD1047 were rapid in onset and readily reversed on washout. SKF10047 inhibited TRPC5 but not TRPM3 or TRPM2. Depletion of Sig1R did not prevent the inhibitory actions of BD1063 or BD1047 and Sig1R did not co-localize with TRPC5 or TRPM3. Conclusions and Implications The data suggest that two types of Sig1R ligand (BD1047/BD1063 and 4-IBP) are inhibitors of receptor- or chemically activated calcium entry channels, acting relatively directly and independently of the Sig1R. Chemical foundations for TRP channel inhibitors are suggested.

Dual-agonist molecules combining glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) activity represent an exciting therapeutic strategy for diabetes treatment. Although challenging due to shared downstream signalling pathways, determining the relative activity of dual agonists at each receptor is essential when developing potential novel therapeutics. The challenge is exacerbated in physiologically relevant cell systems expressing both receptors. To this end, either GIP receptors (GIPR) or GLP-1 receptors (GLP-1R) were ablated via RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonucleases in the INS-1 pancreatic β-cell line. Multiple clonal cell lines harbouring gene disruptions for each receptor were isolated and assayed for receptor activity to identify functional knockouts (KOs). cAMP production in response to GIPR or GLP-1R activation was abolished and GIP- or GLP-1-induced potentiation of glucose-stimulated insulin secretion (GSIS) was attenuated in the cognate KO cell lines. The contributions of individual receptors derived from cAMP and GSIS assays were confirmed in vivo using GLP-1R KO mice in combination with a monoclonal antibody antagonist of GIPR. We have successfully applied CRISPR/Cas9-engineered cell lines to determining selectivity and relative potency contributions of dual-agonist molecules targeting receptors with overlapping native expression profiles and downstream signalling pathways. Specifically, we have characterised molecules as biased towards GIPR or GLP-1R, or with relatively balanced potency in a physiologically relevant β-cell system. This demonstrates the broad utility of CRISPR/Cas9 when applied to native expression systems for the development of drugs that target multiple receptors, particularly where the balance of receptor activity is critical.

Micropropagation and production of Veratrum californicum is most successful when using a premixed Murishage and Skoog basal medium with vitamins and a 5-week subculture cycle at 16 °C for multiplication. These culture conditions provide the best percent survival after acclimatization in the greenhouse. However, clone response to temperature and light quality within culture conditions varies. Micropropagated plants have mass and morphology similar to 2- or 3-year-old seedlings. Acclimatized plantlets can then be grown in the greenhouse using sub-irrigation (ebb and flood) to maintain substrate volumetric water content > 44 %. Growth cycle in the greenhouse must be about 100 days, followed by dormancy for 5 months at 5 °C.

The aim of this study was to generate new insight into chemical regulation of transient receptor potential (TRP) channels with relevance to glucose homeostasis and the metabolic syndrome. Human TRP melastatin 2 (TRPM2), TRPM3, and TRP canonical 5 (TRPC5) were conditionally overexpressed in human embryonic kidney 293 cells and studied by using calcium-measurement and patch-clamp techniques. Rosiglitazone and other peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists were investigated. TRPM2 was unaffected by rosiglitazone at concentrations up to 10 μM but was inhibited completely at higher concentrations (IC(50), ∼22.5 μM). TRPM3 was more potently inhibited, with effects occurring in a biphasic concentration-dependent manner such that there was approximately 20% inhibition at low concentrations (0.1-1 μM) and full inhibition at higher concentrations (IC(50), 5-10 μM). PPAR-γ antagonism by 2-chloro-5-nitrobenzanilide (GW9662) did not prevent inhibition of TRPM3 by rosiglitazone. TRPC5 was strongly stimulated by rosiglitazone at concentrations of ≥10 μM (EC(50), ∼30 μM). Effects on TRPM3 and TRPC5 occurred rapidly and reversibly. Troglitazone and pioglitazone inhibited TRPM3 (IC(50), 12 μM) but lacked effect on TRPC5, suggesting no relevance of PPAR-γ or the thiazolidinedione moiety to rosiglitazone stimulation of TRPC5. A rosiglitazone-related but nonthiazolidinedione PPAR-γ agonist, N-(2-benzoylphenyl)-O-[2-(methyl-2-pyridinylamino)ethyl]-l-tyrosine (GW1929), was a weak stimulator of TRPM3 and TRPC5. The natural PPAR-γ agonist 15-deoxy prostaglandin J(2), had no effect on TRPM3 or TRPC5. The data suggest that rosiglitazone contains chemical moieties that rapidly, strongly, and differentially modulate TRP channels independently of PPAR-γ, potentially contributing to biological consequences of the agent and providing the basis for novel TRP channel pharmacology.

BACKGROUND AND PURPOSE Transient receptor potential canonical 5 (TRPC5) channels are widely expressed, including in the CNS, where they potentiate fear responses. They also contribute to other non-selective cation channels that are stimulated by G-protein-coupled receptor agonists and lipid and redox factors. Steroids are known to modulate fear and anxiety states, and we therefore investigated whether TRPC5 exhibited sensitivity to steroids.

Transient receptor potential canonical 5 (TRPC5) forms cationic channels that are polymodal sensors of factors including oxidized phospholipids, hydrogen peroxide, and reduced thioredoxin. The aim of this study was to expand knowledge of the chemical-sensing capabilities of TRPC5 by investigating dietary antioxidants. Human TRPC5 channels were expressed in HEK 293 cells and studied by patch clamp and intracellular Ca(2+) recording. GFP- and HA-tagged channels were used to quantify plasma membrane localization. Gallic acid and vitamin C suppressed TRPC5 activity if it was evoked by exogenous hydrogen peroxide or lanthanide ions but not by lysophosphatidylcholine or carbachol. Catalase mimicked the effects, suggesting that lanthanide-evoked activity depended on endogenous hydrogen peroxide. Trans-resveratrol, by contrast, inhibited all modes of TRPC5, and its effect was additive with that of vitamin C, suggesting antioxidant-independent action. The IC(50) was ∼10 μM. Diethylstilbestrol, a related hydroxylated stilbene, inhibited TRPC5 with a similar IC(50), but its action contrasted sharply with that of resveratrol in outside-out membrane patches where diethylstilbestrol caused strong and reversible inhibition and resveratrol had no effect, suggesting indirect modulation by resveratrol. Resveratrol did not affect channel surface density, but its effect was calcium-sensitive, indicating an action via a calcium-dependent intermediate. The data suggest previously unrecognized chemical-sensing properties of TRPC5 through multiple mechanisms: (i) inhibition by scavengers of reactive oxygen species because a mode of TRPC5 activity depends on endogenous hydrogen peroxide; (ii) direct channel blockade by diethylstilbestrol; and (iii) indirect, antioxidant-independent inhibition by resveratrol.

BACKGROUND AND PURPOSE The transient receptor potential melastatin-3 (TRPM3) channel forms calcium-permeable, non-selective, cationic channels that are stimulated by pregnenolone sulphate (PregS). Here, we aimed to define chemical requirements of this acute steroid action and potentially reveal novel stimulators with physiological relevance. EXPERIMENTAL APPROACH We used TRPM3 channels over-expressed in HEK 293 cells, with intracellular calcium measurement and whole-cell patch-clamp recording techniques. KEY RESULTS The stimulation of TRPM3 channels was confined to PregS and closely related steroids and not mimicked by other major classes of steroids, including progesterone. Relatively potent stimulation of TRPM3-dependent calcium entry was observed. A sulphate group positioned at ring A was important for strong stimulation but more striking was the requirement for a cis (β) configuration of the side group, revealing previously unrecognized stereo-selectivity and supporting existence of a specific binding site. A cis-oriented side group on ring A was not the only feature necessary for high activity because loss of the double bond in ring B reduced potency and loss of the acetyl group at ring D reduced efficacy and potency. Weak steroid stimulators of TRPM3 channels inhibited effects of PregS, suggesting partial agonism. In silico screening of chemical libraries for non-steroid modulators of TRPM3 channels revealed the importance of the steroid backbone for stimulatory effects. CONCLUSIONS AND IMPLICATIONS Our data defined some of the chemical requirements for acute stimulation of TRPM3 channels by steroids, supporting the existence of a specific and unique steroid binding site. Epipregnanolone sulphate was identified as a novel TRPM3 channel stimulator.

For many ion channels there are no specific pharmacological agents and this impedes searches for the physiological and pathological functions of the ion channels in native systems and restricts opportunity for therapeutic drug development. Antibodies can display high specificity for their target proteins and are routinely engineered to recognise proteins in many experimental procedures and increasingly as therapeutic agents. The unlimited diversity and potential for high specificity make antibodies attractive as biological tools and drugs, although they are not without problems. Along with other investigators we have explored targeting of the E3 extracellular loop (or turret) as an approach for the generation of antibodies with the potential to block certain types of ion channel with isoform specificity. The approach has led to success with nine ion channels and effective use in vitro and in vivo. Here we review current knowledge and experience of channel-blocking antibodies, focusing particularly but not exclusively on E3-targeting.

Multi-well robotic planar patch-clamp has become common in drug development and safety programmes because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favoured method. Here we show the wider potential of the multi-well approach with the capability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints, and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by pre-programmed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 hr depending on the experimental design and yields 16-33 cell recordings.

Background and purpose: Isoform-specific ion channel blockers are useful for target validation in drug discovery and can provide the basis for new therapeutic agents and aid in determination of physiological functions of ion channels. The aim of this study was to generate a specific blocker of human TRPM3 channels as a tool to help investigations of this member of the TRP cationic channel family. Experimental approach: A polyclonal antibody (TM3E3) was made to a conserved peptide of the third extracellular (E3) loop of TRPM3 and tested for binding and functional effect. Studies of channel activity were made by whole-cell planar patch-clamp and fura-2 intracellular Ca2+ measurement. Key results: Ionic current mediated by TRPM3 was inhibited partially by TM3E3 over a period of 5–10 min. Ca2+ entry in TRPM3-expressing cells was also partially inhibited by TM3E3 in a peptide-specific manner and independently of the type of agonist used to activate TRPM3. TM3E3 had no effect on TRPC5, TRPV4, TRPM2 or an endogenous ATP response. Conclusions and implications: The data show the successful development of a specific TRPM3 inhibitor and give further confidence in E3 targeting as an approach to producing isoform-specific ion channel blockers. British Journal of Pharmacology (2008) 155, 567–573; doi:10.1038/bjp.2008.283; published online 7 July 2008

Reducing agents can activate different members of the transient receptor potential (TRP) superfamily of ion channels (TRPC1 and TRPC5) by breaking a disulphide bridge in the extracellular loop adjacent to the ion permation pore. A high concentration of the endogenous reducing agent, thioredoxin, is present in rheumatoid arthritis and may reduce secretion of matrix metalloproteinases by its inhibition of TRPC1 and TRPC5. Mammalian homologues of Drosophila melanogaster transient receptor potential (TRP) are a large family of multimeric cation channels that act, or putatively act, as sensors of one or more chemical factor1,2. Major research objectives are the identification of endogenous activators and the determination of cellular and tissue functions of these channels. Here we show the activation of TRPC5 (canonical TRP 5) homomultimeric and TRPC5–TRPC1 heteromultimeric channels3,4,5 by extracellular reduced thioredoxin, which acts by breaking a disulphide bridge in the predicted extracellular loop adjacent to the ion-selectivity filter of TRPC5. Thioredoxin is an endogenous redox protein with established intracellular functions, but it is also secreted and its extracellular targets are largely unknown6,7,8,9. Particularly high extracellular concentrations of thioredoxin are apparent in rheumatoid arthritis8,10,11,12, an inflammatory joint disease that disables millions of people worldwide13. We show that TRPC5 and TRPC1 are expressed in secretory fibroblast-like synoviocytes from patients with rheumatoid arthritis, that endogenous TRPC5–TRPC1 channels of the cells are activated by reduced thioredoxin, and that blockade of the channels enhances secretory activity and prevents the suppression of secretion by thioredoxin. The data indicate the presence of a previously unrecognized ion-channel activation mechanism that couples extracellular thioredoxin to cell function.

In a screen of potential lipid regulators of transient receptor potential (TRP) channels, we identified sphingosine-1–phosphate (S1P) as an activator of TRPC5. We explored the relevance to vascular biology because S1P is a key cardiovascular signaling molecule. TRPC5 is expressed in smooth muscle cells of human vein along with TRPC1, which forms a complex with TRPC5. Importantly, S1P also activates the TRPC5–TRPC1 heteromultimeric channel. Because TRPC channels are linked to neuronal growth cone extension, we considered a related concept for smooth muscle. We find S1P stimulates smooth muscle cell motility, and that this is inhibited by E3-targeted anti-TRPC5 antibody. Ion permeation involving TRPC5 is crucial because S1P-evoked motility is also suppressed by the channel blocker 2-aminoethoxydiphenyl borate or a TRPC5 ion-pore mutant. S1P acts on TRPC5 via two mechanisms, one extracellular and one intracellular, consistent with its bipolar signaling functions. The extracellular effect appears to have a primary role in S1P-evoked cell motility. The data suggest S1P sensing by TRPC5 calcium channel is a mechanism contributing to vascular smooth muscle adaptation.

Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.

TRPC calcium channels are emerging as a ubiquitous feature of vertebrate cells, but understanding of them is hampered by limited knowledge of the mechanisms of activation and identity of endogenous regulators. We have revealed that one of the TRPC channels, TRPC5, is strongly activated by common endogenous lysophospholipids including lysophosphatidylcholine (LPC) but, by contrast, not arachidonic acid. Although TRPC5 was stimulated by agonists at G-protein-coupled receptors, TRPC5 activation by LPC occurred downstream and independently of G-protein signaling. The effect was not due to the generation of reactive oxygen species or because of a detergent effect of LPC. LPC activated TRPC5 when applied to excised membrane patches and thus has a relatively direct action on the channel structure, either because of a phospholipid binding site on the channel or because of sensitivity of the channel to perturbation of the bilayer by certain lipids. Activation showed dependence on side-chain length and the chemical head-group. The data revealed a previously unrecognized lysophospholipid-sensing capability of TRPC5 that confers the property of a lipid ionotropic receptor.

Biologics represent a fast-growing class of therapeutics in the pharmaceutical sector. Discovery of therapeutic antibodies and characterization of peptides can necessitate high expression of the target gene requiring the generation of clonal stably transfected cell lines. Traditional challenges of stable cell line transfection include gene silencing and cell-to-cell variability. Our inability to control these can present challenges in lead isolation. Recent progress in site-specific targeting of transgene to specific genomic loci has transformed the ability to generate stably transfected mammalian cell lines. In this article, we describe how the use of the Jump-In platform (Life Technologies, Carlsbad, CA) has been applied to drug discovery projects. It can easily and rapidly generate homogeneous high-expressing cell pools with a high degree of reproducibility. Their use in cell-based screening to identify specific binders, identify binding to relevant species variants, or detect functionally relevant therapeutic antibodies is central in driving drug discovery.

There is demand for isoform-specific ion channel inhibitors as tools to investigate the biology of -endogenous ion channels and validate them as targets in drug discovery programs. There is also hope that such inhibitors may be new therapeutic agents or provide the foundation for such agents. However, in practice, it is commonly experienced that inhibitors lack sufficient specificity, fail to distinguish between members of a class of ion channel, or have other (non-ion channel) off-target effects. Due to their extraordinary specificity, antibodies offer a potentially attractive strategy for overcoming these problems. Inhibitory antibodies acting at the extracellular face of ion channels are particularly attractive because there is enhanced possibility for specificity and intracellular delivery methods are not required. Here we describe experience with such an antibody approach and methodology for generating agents based on anti-peptide polyclonal antibodies.

There is demand for isoform-specific ion channel inhibitors as tools to investigate the biology of -endogenous ion channels and validate them as targets in drug discovery programs. There is also hope that such inhibitors may be new therapeutic agents or provide the foundation for such agents. However, in practice, it is commonly experienced that inhibitors lack sufficient specificity, fail to distinguish between members of a class of ion channel, or have other (non-ion channel) off-target effects. Due to their extraordinary specificity, antibodies offer a potentially attractive strategy for overcoming these problems. Inhibitory antibodies acting at the extracellular face of ion channels are particularly attractive because there is enhanced possibility for specificity and intracellular delivery methods are not required. Here we describe experience with such an antibody approach and methodology for generating agents based on anti-peptide polyclonal antibodies.

Background and Purpose The Sigma-1 receptor (Sig1R) impacts on calcium ion signalling and has a plethora of ligands. This study investigated Sig1R and its ligands in relation to endogenous calcium events of endothelial cells and transient receptor potential (TRP) channels. Experimental Approach Intracellular calcium and patch clamp measurements were made from human saphenous vein endothelial cells and HEK 293 cells expressing exogenous human TRPC5, TRPM2 or TRPM3. Sig1R ligands were applied and short interfering RNA was used to deplete Sig1R. TRP channels tagged with fluorescent proteins were used for subcellular localization studies. Key Results In endothelial cells, 10–100 μM of the Sig1R antagonist BD1063 inhibited sustained but not transient calcium responses evoked by histamine. The Sig1R agonist 4-IBP and related antagonist BD1047 were also inhibitory. The Sig1R agonist SKF10047 had no effect. Sustained calcium entry evoked by VEGF or hydrogen peroxide was also inhibited by BD1063, BD1047 or 4-IBP, but not SKF10047. 4-IBP, BD1047 and BD1063 inhibited TRPC5 or TRPM3, but not TRPM2. Inhibitory effects of BD1047 were rapid in onset and readily reversed on washout. SKF10047 inhibited TRPC5 but not TRPM3 or TRPM2. Depletion of Sig1R did not prevent the inhibitory actions of BD1063 or BD1047 and Sig1R did not co-localize with TRPC5 or TRPM3. Conclusions and Implications The data suggest that two types of Sig1R ligand (BD1047/BD1063 and 4-IBP) are inhibitors of receptor- or chemically activated calcium entry channels, acting relatively directly and independently of the Sig1R. Chemical foundations for TRP channel inhibitors are suggested.

Transient Receptor Potential Melastatin 3 (TRPM3) is a widely expressed calcium-permeable non-selective cation channel that is stimulated by high concentrations of nifedipine or by physiological steroids that include pregnenolone sulphate. Here we sought to identify steroids that inhibit TRPM3. Channel activity was studied using calcium-measurement and patch-clamp techniques. Progesterone (0.01–10μM) suppressed TRPM3 activity evoked by pregnenolone sulphate. Progesterone metabolites and 17β-oestradiol were also inhibitory but the effects were relatively small. Dihydrotestosterone was an inhibitor at concentrations higher than 1μM. Corticosteroids lacked effect. Overlay assays indicated that pregnenolone sulphate, progesterone and dihydrotestosterone bound to TRPM3. In contrast to dihydrotestosterone, progesterone inhibited nifedipine-evoked TRPM3 activity or activity in the absence of an exogenous activator, suggesting a pregnenolone sulphate-independent mechanism of action. Dihydrotestosterone, like a non-steroid look-alike compound, acted as a competitive antagonist at the pregnenolone sulphate binding site. Progesterone inhibited endogenous TRPM3 in vascular smooth muscle cells. Relevance of TRPM3 or the progesterone effect to ovarian cells, which have been suggested to express TRPM3, was not identified. The data further define a chemical framework for competition with pregnenolone sulphate at TRPM3 and expand knowledge of steroid interactions with TRPM3, suggesting direct steroid binding and pregnenolone sulphate-independent inhibition by progesterone.

Background Calcium-permeable channels are known to have roles in many mammalian cell types but the expression and contribution of such ion channels in synovial cells is mostly unknown. The objective of this study was to investigate the potential relevance of Transient Receptor Potential Melastatin 3 (TRPM3) channel to fibroblast-like synoviocytes (FLSs) of patients with rheumatoid arthritis. Methods The study used RT-PCR and immunofluorescence to detect mRNA and protein. Intracellular calcium measurement detected channel activity in a FLS cell-line and primary cultures of FLSs from patients with rheumatoid arthritis. Enzyme-linked immunosorbent assays measured hyaluronan. Results Endogenous expression of TRPM3 was detected. Previously reported stimulators of TRPM3 sphingosine and pregnenolone sulphate evoked sustained elevation of intracellular calcium in FLSs. The FLS cell-line showed an initial transient response to sphingosine which may be explained by TRPV4 channels but was not observed in FLSs from patients. Blocking antibody targeted to TRPM3 inhibited sustained sphingosine and pregnenolone sulphate responses. Secretion of hyaluronan, which contributes adversely in rheumatoid arthritis, was suppressed by pregnenolone sulphate in FLSs from patients and the effect was blocked by anti-TRPM3 antibody. Conclusions The data suggest that FLSs of patients with rheumatoid arthritis express TRPM3-containing ion channels that couple negatively to hyaluronan secretion and can be stimulated by pharmacological concentrations of pregnenolone sulphate.

Rationale : Transient receptor potential melastatin (TRPM)3 is a calcium-permeable ion channel activated by the neurosteroid pregnenolone sulfate and positively coupled to insulin secretion in β cells. Although vascular TRPM3 mRNA has been reported, there is no knowledge of TRPM3 protein or its regulation and function in the cardiovascular system. Objective : To determine the relevance and regulation of TRPM3 in vascular biology. Methods and Results : TRPM3 expression was detected at mRNA and protein levels in contractile and proliferating vascular smooth muscle cells. Calcium entry evoked by pregnenolone sulfate or sphingosine was suppressed by TRPM3 blocking antibody or knock-down of TRPM3 by RNA interference. Low-level constitutive TRPM3 activity was also detected. In proliferating cells, channel activity was coupled negatively to interleukin-6 secretion via a calcium-dependent mechanism. In freshly isolated aorta, TRPM3 positively modulated contractile responses independently of L-type calcium channels. Concentrations of pregnenolone sulfate required to evoke responses were higher than the known plasma concentrations of the steroids, leading to a screen for other stimulators. β-Cyclodextrin was one of few stimulators of TRPM3, revealing the channels to be partially suppressed by endogenous cholesterol, the precursor of pregnenolone. Elevation of cholesterol further suppressed channel activity and loading with cholesterol to generate foam cells precluded observation of TRPM3 activity. Conclusions : The data suggest functional relevance of TRPM3 in contractile and proliferating phenotypes of vascular smooth muscle cells, significance of constitutive channel activity, regulation by cholesterol, and potential value of pregnenolone sulfate in therapeutic vascular modulation.

The Transient Receptor Potential (TRP) channels form a diverse superfamily of cation channels. Currently there are few well characterised, selective, physiological regulators of TRP channels, which can impede the discovery of native channel functions. TRPM3, a member of the melastatin subfamily of TRP channels, is reported to form constitutively active Ca2+-permeable cation channels activated by sphingolipids, hypotonic shock, and the neurosteroid pregnenolone sulphate. Expression is predominantly in brain and kidney, although expression has recently been reported in pancreatic β-cells. Here we report TRPM3 expression and function in vascular smooth muscle cells. Anti-TRPM3 blocking antibody and short-interfering RNA targeted to TRPM3 were used to confirm the involvement of TRPM3 in the pregnenolone sulphate elicited Ca2+ influx in cultured human vascular smooth muscle cells. Although pregnenolone sulphate is a useful pharmacological and potentially therapeutic agent, the physiological significance in the vasculature is unknown. We therefore screened for novel modulators, and found that cholesterol inhibited both pregnenolone sulphate-induced and constitutive TRPM3 activity. The data suggest TRPM3 is a novel calcium-entry channel of vascular smooth muscle cells.The research was supported by a BBSRC Industrial Co-operative Award in Science and Engineering, the Wellcome Trust and the British Heart Foundation.