Decisional conflict among couples seeking specialty treatment for infertility in the USA: a longitudinal exploratory study.

STUDY QUESTION: What are couples' decisional conflicts around family-building approaches before and after seeking a specialty consultation for infertility? SUMMARY ANSWER: Decisional conflict is high among couples before an initial specialty consultation for infertility; on average, women resolved decisional conflict more quickly than men. WHAT IS KNOWN ALREADY: Couples have multiple options for addressing infertility, and decisional conflict may arise due to lack of information, uncertainty about options and potential risks or challenges to personal values. STUDY DESIGN, SIZE, DURATION: We conducted a total of 385 interviews and 405 surveys for this longitudinal, mixed-methods cohort study of 34 opposite-sex couples who sought a new reproductive specialty consultation (n = 68), who enrolled before the initial consultation and were followed over 12 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: The in-depth, semi-structured interviews included questions about information gathering, deliberation and decision-making, and self-administered surveys included the Decisional Conflict Scale (DCS), at six time points over 12 months. A DCS total score of 25 is associated with implementing a decision, and higher scores indicate more decisional conflict. A systematic content analysis of interview transcripts identified major themes. Paired t tests identified differences in DCS between women and men within couples. Linear mixed models predicted changes in DCS over time, adjusting for sociodemographic and fertility-related factors. MAIN RESULTS AND THE ROLE OF CHANCE: The major qualitative themes were communication with partners, feeling supported and/or pressured in decision (s), changing decisions over time and ability to execute a desired decision. Average DCS scores were highest before the initial consultation. Within couples, men had significantly higher decisional conflict than women pre-consultation (48.9 versus 40.2, P = 0.037) and at 2 months (28.9 versus 22.1, P = 0.015), but differences at other time points were not significant. In adjusted models, predicted DCS scores declined over time, with women, on average, reaching the DCS threshold for implementing a decision at 2 months while for men it was not until 4 months. LIMITATIONS, REASONS FOR CAUTION: This is a convenience sample from a single center, and generalizability may be limited. WIDER IMPLICATIONS OF THE FINDINGS: Understanding how couples discuss and make decisions regarding family-building could improve the delivery of patient-centered infertility care. Our findings are the first to prospectively explore decisional conflict at multiple time points in both men and women; the observed gender differences underlie the importance of supporting both partners in clinical decision-making for infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Institute of Child Health and Human Development under Grant [R21HD071332], the Research and Education Program Fund, of the Advancing a Healthier Wisconsin endowment at Medical College of Wisconsin, the National Research Service Award under Grant [T32 HP10030] and the use of REDCap for data collection from the National Center for Advancing Translational Sciences, National Institutes of Health under Grant through [8UL1TR000055]. The authors have no competing interests.

Pool-BCGA: a parallelised generation-free genetic algorithm for the ab initio global optimisation of nanoalloy clusters.

The Birmingham cluster genetic algorithm is a package that performs global optimisations for homo- and bimetallic clusters based on either first principles methods or empirical potentials. Here, we present a new parallel implementation of the code which employs a pool strategy in order to eliminate sequential steps and significantly improve performance. The new approach meets all requirements of an evolutionary algorithm and contains the main features of the previous implementation. The performance of the pool genetic algorithm is tested using the Gupta potential for the global optimisation of the Au10Pd10 cluster, which demonstrates the high efficiency of the method. The new implementation is also used for the global optimisation of the Au10 and Au20 clusters directly at the density functional theory level.

Platelet-derived growth factors and fibroblast growth factors are mitogens for rat Schwann cells.

Rat sciatic nerve Schwann cells in culture respond to a limited range of mitogens, including glial growth factor, transforming growth factors beta-1 and beta-2 (TGF-beta 1, TGF-beta 2), some cell membrane-associated factors, and to agents such as cholera toxin and forskolin which raise intracellular levels of cAMP. These responses require the presence of FCS, which exhibits little or no mitogenic activity in the absence of other factors. However, we recently found that forskolin greatly potentiates the mitogenic signal from TGFs-beta 1 and beta 2, raising the possibility that cAMP might couple other factors to mitogenesis. We have therefore screened a range of candidate mitogens using DNA synthesis assays. Other than TGFs-beta and glial growth factor, none of the factors tested were mitogenic in the presence of 10% serum alone. With the addition of forskolin, however, porcine PDGF, human PDGF, acidic and basic FGF were potent mitogens for rat Schwann cells, stimulating DNA synthesis and increasing cell number. Cholera toxin and dibutyrylcyclicAMP, but not 1,9-dideoxyforskolin, can substitute for forskolin indicating that the mitogenic effect is mediated via adenylyl cyclase activation. Porcine PDGF gave half-maximal stimulation at 15 pM, and human PGDF an equivalent response at 1 nM. Basic FGF was half maximal at 5 pM, acidic FGF at 1 nM. The recognition of PDGFs and FGFs as mitogens for Schwann cells has many implications for the study of Schwann cell proliferation in the development and regeneration of nerves, and in Schwann cell tumorigenesis.

Transforming growth factors-beta 1 and beta 2 are mitogens for rat Schwann cells.

Transforming growth factor-beta 1 (TGF-beta 1) and TGF-beta 2 were found to be potent mitogens for purified rat Schwann cells, each stimulating DNA synthesis in quiescent cells and also increasing their proliferation rate. Half-maximal stimulation of DNA synthesis occurred at approximately 0.1 ng/ml TGF-beta 1 or TGF-beta 2. Mitogenic stimulation by TGF-beta 1 and TGF-beta 2 was enhanced by forskolin, which activates adenylate cyclase, at concentrations up to 0.5 microM forskolin. However, at 5 microM forskolin, the synergistic interaction between forskolin and TGF-beta 1 was abolished. These results are in contrast to the observed synergy between forskolin and another Schwann cell mitogen, glial growth factor (GGF). Both 0.5 and 5 microM forskolin were found to enhance the stimulation of DNA synthesis by partially purified GGF (GGF-CM). As well as being functionally distinct, TGF-beta 1 and GGF-CM activities were also physically separable by chromatography on a Superose 12 gel permeation column. Thus, TGF-beta 1 and beta 2 are rat Schwann cell mitogens, and Schwann cells are one of the few normal cell populations to respond mitogenically to TGF-beta.

Ethanol elicits and potentiates nociceptor responses via the vanilloid receptor-1.

The vanilloid receptor-1 (VR1) is a heat-gated ion channel that is responsible for the burning sensation elicited by capsaicin. A similar sensation is reported by patients with esophagitis when they consume alcoholic beverages or are administered alcohol by injection as a medical treatment. We report here that ethanol activates primary sensory neurons, resulting in neuropeptide release or plasma extravasation in the esophagus, spinal cord or skin. Sensory neurons from trigeminal or dorsal root ganglia as well as VR1-expressing HEK293 cells responded to ethanol in a concentration-dependent and capsazepine-sensitive fashion. Ethanol potentiated the response of VR1 to capsaicin, protons and heat and lowered the threshold for heat activation of VR1 from approximately 42 degrees C to approximately 34 degrees C. This provides a likely mechanistic explanation for the ethanol-induced sensory responses that occur at body temperature and for the sensitivity of inflamed tissues to ethanol, such as might be found in esophagitis, neuralgia or wounds.

The effect of neurotrophic factors on morphology, TRPV1 expression and capsaicin responses of cultured human DRG sensory neurons.

We have studied the effect of key neurotrophic factors (NTFs) on morphology, levels of the vanilloid receptor-1 (TRPV1) and responses to capsaicin in adult human sensory neurons in vitro. Avulsed dorsal root ganglia (DRG, n = 5) were cultured with or without a combination of nerve growth factor (NGF), glial cell (line)-derived growth factor (GDNF) and neurotrophin3 (NT3) for 5 days. In the absence of NTFs, the diameter of neurons ranged from 20 to 100 microm (mean 42 +/- 4 microm). Adding NTFs caused a significant increase in neuronal sizes, up to 120 microm (mean diameter 62 +/- 5 microm, P < 0.01, t-test), an overall 35% increase of TRPV1-positive neurons (P < 0.003), and notably of large TRPV1-positive neurons > 80 microm (P < 0.05). Responses to capsaicin were significantly enhanced with calcium ratiometry (P < 0.0001). Short duration (1h) exposure of dissociated sensory neurons to NTFs increased numbers of TRPV1-positive neurons, but not of TRPV3, Nav 1.8 and IK1 and the morphological size-distribution remained similar to intact post-mortem DRG neurons. NTFs thus increase size, elevate TRPV1 levels and enhance capsaicin responses in cultured human DRG neurons; these changes may relate to pathophysiology in disease states, and provide an in vitro model to study novel analgesics.

Microfluidic solid phase suspension transport with an elastomer-based, single piezo-actuator, micro throttle pump.

We report a Micro Throttle Pump (MTP) which has been shown to pump 5 microm diameter polystyrene beads at a concentration of 4.5 x 10(7) beads ml(-1). This new MTP design is constructed in a straightforward manner and actuated by a single piezoelectric (PZT) element. Maximum flow rates at 800 Hz drive frequency of 132 microl min(-1) with water and 108 microl min(-1) with a bead suspension were obtained. Maximum back-pressures of 6 kPa were observed in both cases. The reported MTP employs specific location of distinct internal microfluid structures cast in a single compliant elastomeric substrate to exploit the opposing directions of flexure of regions of a piezoelectric-glass composite bonded to the elastomer. By this novel means, distinct flexural regions, exhibiting compressive and tensile stresses respectively, allow both the pump's integrated input and output throttles and its pump chamber to be actuated concurrently by a single PZT. To support MTP design we also report the characterisation of an individual throttle's resistance as a function of actuator deflection and discuss the underlying mechanism of the throttling effect.

Vagal selective effects of ruthenium red on the jejunal afferent fibre response to ischaemia in the rat.

A variety of inflammatory mediators and local metabolites, have been implicated in the sensitivity of intestinal afferent fibres to brief periods of ischaemia and reperfusion. As yet, the contribution of the vanilloid transient receptor potential (TRPV)1 receptor to the response to intestinal ischaemia remains undetermined. In the present study, the effect of pretreatment with the competitive TRPV1 antagonist capsazepine and the non-selective TRPV channel antagonist ruthenium red, on the mesenteric afferent fibre response to ischaemia was examined. In control animals there was a reproducible biphasic increase in whole nerve afferent fibre activity during two brief periods of ischaemia. Treatment with ruthenium red significantly attenuated the early phase increase in afferent fibre activity during ischaemia. However, capsazepine treatment did not significantly alter the afferent fibre response to either ischaemia or reperfusion. Further experiments in chronically vagotomized animals indicated that the early phase response to ischaemia was mediated via vagal afferent fibres. The mechanism via which ruthenium red selectively inhibited vagal afferent fibres during ischaemia is unknown, but it does not appear to involve blockade of the TRPV1 receptor.

The quality assurance programme of the Radiotherapy Group of the European Organisation for Research and Treatment of Cancer: past, present and future.

As early as in 1982, the European Organisation for Research and Treatment of Cancer Radiotherapy Group established a quality assurance programme. In the course of 20 years, quality assurance procedures have become a vast and important part of the activities of the group. Today, the membership committee uses standard procedures based on minimal requirements to evaluate current members and new membership applications. Moreover, for every new trial, specific quality assurance procedures are an integral part of the preparation of the protocol and executed under the responsibility of the study coordinator. With the growing complexity of the radiotherapy techniques used in the framework of the more recent trials, quality assurance procedures have also become more complex including trial specific phantom based measurements. Future ways to evaluate all steps of the radiotherapy process using a common platform connecting all users with the internet are currently under development.

Palmitoylethanolamide enhances anandamide stimulation of human vanilloid VR1 receptors.

In human embryonic kidney cells over-expressing the human vanilloid receptor type 1 (VR1), palmitoylethanolamide (PEA, 0.5-10 microM) enhanced the effect of arachidonoylethanolamide (AEA, 50 nM) on the VR1-mediated increase of the intracellular Ca2+ concentration. PEA (5 microM) decreased the AEA half-maximal concentration for this effect from 0.44 to 0.22 microM. The PEA effect was not due to inhibition of AEA hydrolysis or adhesion to non-specific sites, since bovine serum albumin (0.01-0.25%) potently inhibited AEA activity, and PEA also enhanced the effect of low concentrations of the VR1 agonists resiniferatoxin and capsaicin. PEA (5 microM) enhanced the affinity of AEA for VR1 receptors as assessed in specific binding assays. These data suggest that PEA might be an endogenous enhancer of VR1-mediated AEA actions.

Overlap between the ligand recognition properties of the anandamide transporter and the VR1 vanilloid receptor: inhibitors of anandamide uptake with negligible capsaicin-like activity.

Some synthetic agonists of the VR1 vanilloid (capsaicin) receptor also inhibit the facilitated transport into cells of the endogenous cannabinoid anandamide (arachidonoylethanolamide, AEA). Here we tested several AEA derivatives containing various derivatized phenyl groups or different alkyl chains as either inhibitors of the AEA membrane transporter (AMT) in intact cells or functional agonists of the VR1 vanilloid receptor in HEK cells transfected with the human VR1. We found that four known AMT inhibitors, AM404, arvanil, olvanil and linvanil, activate VR1 receptors at concentrations 400-10000-fold lower than those necessary to inhibit the AMT. However, we also found three novel AEA derivatives, named VDM11, VDM12 and VDM13, which inhibit the AMT as potently as AM404 but exhibit little or no agonist activity at hVR1. These compounds are weak inhibitors of AEA enzymatic hydrolysis and poor CB(1)/CB(2) receptor ligands. We show for the first time that, despite the overlap between the chemical moieties of AMT inhibitors and VR1 agonists, selective inhibitors of AEA uptake that do not activate VR1 (e.g. VDM11) can be developed.

Clinical impact of dosimetry quality assurance programmes assessed by radiobiological modelling of data from the thermoluminescent dosimetry study of the European Organization for Research and Treatment of Cancer.

The European Organization for Research and Treatment of Cancer (EORTC) Radiotherapy Group initiated its mailed thermoluminescence dosimetry (TLD) programme in 1986. The aim of the present study was to evaluate the clinical relevance of variations in beam output detected in the period 1993 to 1996. A total of 140 beam outputs were checked (26 for cobalt-60 units and 114 for linear accelerators) in 35 centres. Clinical dose-response data for tumour control and normal tissue morbidity were used to assess the variation in clinical outcome resulting from variability in beam output. For 75 checked beams with nominal accelerating potentials (n. a.p.) of 6 MV or less the mean ratio, +/- standard deviation (S.D.) of measured to stated output was 1.004+/-0.020. For 65 beams with n. a.p. of 8 MV or more, the ratio was 1.009+/-0.021. Even with this relatively high level of precision, broad distributions of estimated tumour control or normal tissue morbidity were found. In the 10% of the beams with the most pronounced underdosage, the loss in tumour control probability was estimated at 7-8 percentage points. Likewise, in the 10% of the beams with the most pronounced overdosage, the increase in mild/moderate morbidity was 19-22 percentage points. For severe morbidity the same beams raised the estimated incidence of severe complications from 5% to 9-10%. An estimation of the loss of uncomplicated cure probability was about 1% for both high and low energy beams. Sequential mailings considerably improved the uniformity of clinical outcome. We conclude that small deviations in beam output may lead to clinically important variations in outcome. Substantial reductions in the variation between measured and stated output can be achieved by sequential mailings. Mailed TLD checks should be an integral part of a continuously ongoing quality assurance activity in radiotherapy.

Quality assurance of EORTC trial 22922/10925 investigating the role of internal mammary--medial supraclavicular irradiation in stage I-III breast cancer: the individual case review.

To assess consistency among participants in an European Organisation for Research and Treatment of Cancer (EORTC) phase III trial randomising between irradiation and no irradiation of the internal mammary and medial supraclavicular (IM-MS) lymph nodes, all participating institutes were invited to send data from 3 patients in each arm as soon as they started accrual. The evaluation focused on eligibility, compliance with the radiotherapy guidelines, treatment techniques and dose prescription to the IM-MS region. Nineteen radiotherapy departments provided a total of 111 cases, all being eligible. Minor discrepancies were found in the surgery and pathology data in almost half the patients. Major radiotherapy protocol deviations were very limited: 2 cases of unwarranted irradiation of the supraclavicular region and a significant dose deviation to the internal mammary region in 5 patients. The most frequently observed minor protocol deviation was the absence of delineation of the target volumes in 80% of the patients. By detecting systematic protocol deviations in an early phase of the trial, recommendations made to all the participating institutes should improve the interinstitutional consistency and promote a high-quality treatment.

Macrophages modify beta-VLDL by proteolysis and enhance subsequent lipid accumulation in arterial smooth muscle cells.

Murine resident peritoneal macrophages (MRPM), incubated with beta-very low density lipoprotein (beta-VLDL), modify the beta-VLDL, producing an increase in the mobility of the lipoprotein. The modification does not result in an increase of thiobarbituric acid-reactive substances (TBARS) in the lipoprotein, and is not inhibited by butylated hydroxyanisole (BHA), EDTA, removal of copper and iron from the medium, or by diphenyliodonium (DPI), suggesting that the mechanism of modification is independent of oxidation. Macrophage conditioned medium performed the modification in the absence of cells, and phenylmethylsulphonyl fluoride (PMSF) inhibited beta-VLDL modification, whereas other protease inhibitors did not, suggesting that a secreted neutral serine protease may possibly be involved in the mechanism. The modified beta-VLDL enhanced the accumulation of cholesterol esters by smooth muscle cells (SMC).

A comparison of dose distributions of proton and photon beams in stereotactic conformal radiotherapy of brain lesions.

PURPOSE: Micromultileaf collimators (mMLC) have recently been introduced to conform photon beams in stereotactic irradiation of brain lesions. Proton beams and stereotactic conformal radiotherapy (SCRT) can be used to tailor the dose to nonspherical targets, as most tumors of the brain are irregularly shaped. Comparative planning of brain lesions using either proton or stereotactically guided photon beams was done to assess the institution's clinically available modality for three-dimensional conformal radiotherapy. METHODS AND MATERIALS: For the photon treatment, multiple stereotactically guided uniform intensity beams from a linear accelerator were used, each conformed to a projection of the planning target volume (PTV) by a mMLC. Proton beams were delivered from an isocentrically mounted gantry, using the spot-scanning technique and energy modulation. Seven patients were scanned in a stereotactic frame; target volumes and organs at risk (OAR) were delineated with the help of MR images. Four different lesions were selected: (1) concave, (2) ellipsoid isolated, (3) superficial and close to an organ at risk, and (4) irregular complex. Dose distributions in the PTV and critical structures were calculated using three-dimensional treatment-planning systems, followed by both a quantitative (by dose--volume histogram and conformity index) and qualitative (visual inspection) assessment of the plans. RESULTS: A high degree of conformation was achieved with a mMLC and stereotactic uniform intensity beams with comparable conformity indices to protons for 5 out of 7 plans, especially for superficial or spherical lesions. In the cases studied, the conformity index was better for protons than for photons for complex or concave lesions, or when the PTV was in the neighborhood of critical structures. CONCLUSION: The results for the cases studied, show that for simple geometries or for superficial lesions, there is no advantage in using protons. However, for complex PTV shapes, or when the PTV is in the vicinity of critical structures, protons seem to be potentially better than the fixed-field photon technique.

The potential impact of treatment variations on the results of radiotherapy of the internal mammary lymph node chain: a quality-assurance report on the dummy run of EORTC Phase III randomized trial 22922/10925 in Stage I--III breast cancer(1).

PURPOSE: To present the results of the dummy run of the European Organization for Research and Treatment of Cancer (EORTC) trial investigating the role of adjuvant internal mammary and medial supraclavicular (IM-MS) irradiation in Stage I--III breast cancer. METHODS AND MATERIALS: All participating institutions were asked to produce a treatment plan without (Arm 1) and with (Arm 2) simultaneous IM-MS irradiation of 1 patient after mastectomy and of 1 patient after lumpectomy. Thirty-two dummy runs have been evaluated for compliance to protocol guidelines, with respect to treatment technique and dose prescription. RESULTS: A number of more or less important deviations in treatment setup and prescription have been found. The dose in the IM-MS region deviated significantly from the prescribed dose in 10% of the cases for Arm 1, and in 21% for Arm 2. Assuming a true 5% 10-year survival benefit from optimal IM-MS irradiation, an increase of only 3.8% will be found due to this suboptimal dose distribution. CONCLUSION: In the dummy run, a number of potential systematic protocol deviations that might lead to false-negative results were detected. By providing recommendations to the participating institutions, we expect to improve the interinstitutional consistency and to promote a high quality irradiation in all institutions participating in the trial.

Vanilloid and TRP channels: a family of lipid-gated cation channels.

The emergence of the TRP (C) and vanilloid (TRPV) receptor family of Ca(2+) permeable channels has started to provide molecular focus to a linked group of ion channels whose common feature is activation primarily by intracellular ligands. These channels have a central role in Ca(2+) homeostasis in virtually all cells and in particular those that lack voltage-gated Ca(2+) channels. We will discuss recent work that is more precisely defining both molecular form and physiological function of this important group of Ca(2+) permeable channels with particular focus on the intracellular ligands that gate and modulate channel activity.

Identification and characterisation of SB-366791, a potent and selective vanilloid receptor (VR1/TRPV1) antagonist.

Vanilloid receptor-1 (TRPV1) is a non-selective cation channel, predominantly expressed by peripheral sensory neurones, which is known to play a key role in the detection of noxious painful stimuli, such as capsaicin, acid and heat. To date, a number of antagonists have been used to study the physiological role of TRPV1; however, antagonists such as capsazepine are somewhat compromised by non-selective actions at other receptors and apparent modality-specific properties. SB-366791 is a novel, potent, and selective, cinnamide TRPV1 antagonist isolated via high-throughput screening of a large chemical library. In a FLIPR-based Ca(2+)-assay, SB-366791 produced a concentration-dependent inhibition of the response to capsaicin with an apparent pK(b) of 7.74 +/- 0.08. Schild analysis indicated a competitive mechanism of action with a pA2 of 7.71. In electrophysiological experiments, SB-366791 was demonstrated to be an effective antagonist of hTRPV1 when activated by different modalities, such as capsaicin, acid or noxious heat (50 degrees C). Unlike capsazepine, SB-366791 was also an effective antagonist vs. the acid-mediated activation of rTRPV1. With the aim of defining a useful tool compound, we also profiled SB-366791 in a wide range of selectivity assays. SB-366791 had a good selectivity profile exhibiting little or no effect in a panel of 47 binding assays (containing a wide range of G-protein-coupled receptors and ion channels) and a number of electrophysiological assays including hippocampal synaptic transmission and action potential firing of locus coeruleus or dorsal raphe neurones. Furthermore, unlike capsazepine, SB-366791 had no effect on either the hyperpolarisation-activated current (I(h)) or Voltage-gated Ca(2+)-channels (VGCC) in cultured rodent sensory neurones. In summary, SB-366791 is a new TRPV1 antagonist with high potency and an improved selectivity profile with respect to other commonly used TRPV1 antagonists. SB-366791 may therefore prove to be a useful tool to further study the biology of TRPV1.

Characterization using FLIPR of rat vanilloid receptor (rVR1) pharmacology.

The vanilloid receptor (VR1) is a ligand-gated ion channel, which plays an important role in nociceptive processing. Therefore, a pharmacological characterization of the recently cloned rat VR1 (rVR1) was undertaken. HEK293 cells stable expressing rVR1 (rVR1-HEK293) were loaded with Fluo-3AM and then incubated at 25 degrees C for 30 min with or without various antagonists or signal transduction modifying agents. Then intracellular calcium concentrations ([Ca(2+)](i)) were monitored using FLIPR, before and after the addition of various agonists. The rank order of potency of agonists (resiniferatoxin (RTX)>capsaicin>olvanil>PPAHV) was as expected, and all were full agonists. The potencies of capsaicin and olvanil, but not RTX or PPAHV, were enhanced at pH 6.4 (pEC(50) values of 7.47+/-0.06, 7.16+/-0.06, 8.19+/-0.06 and 6.02+/-0.03 respectively at pH 7.4 vs 7.71+/-0.05, 7.58+/-0.14, 8.10+/-0.05 and 6.04+/-0.08 at pH 6.4). Capsazepine, isovelleral and ruthenium red all inhibited the capsaicin (100 nM)-induced Ca(2+) response in rVR1-HEK293 cells, with pK(B) values of 7.52+/-0.08, 6.92+/-0.11 and 8.09+/-0.12 respectively (n=6 each). The response to RTX and olvanil were also inhibited by these compounds. None displayed any agonist-like activity. The removal of extracellular Ca(2+) abolished, whilst inhibition of protein kinase C with chelerythrine chloride (10 microM) partially (approximately 20%) inhibited, the capsaicin (10 microM)-induced Ca(2+) response. However, tetrodotoxin (3 microM), nimodipine (10 microM), omega-GVIA conotoxin (1 microM), thapsigargin (1 microM), U73122 (3 microM) or H-89 (3 microM) had no effect on the capsaicin (100 nM)-induced response. In conclusion, the recombinant rVR1 stably expressed in HEK293 cells acts as a ligand-gated Ca(2+) channel with the appropriate agonist and antagonist pharmacology, and therefore is a suitable model for studying the effects of drugs at this receptor.

The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1).

The endogenous cannabinoid anandamide was identified as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR-based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 microM) and capsaicin (1 microM) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 microM). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC(50) values of 5. 94+/-0.06 (n=5) and 7.13+/-0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pK(B) of 7.40+/-0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide-induced calcium response and vice versa. In conclusion, this study has demonstrated for the first time that anandamide acts as a full agonist at the human VR1.