Use of a fluorescent imaging plate reader--based calcium assay to assess pharmacological differences between the human and rat vanilloid receptor

The cloned vanilloid receptor 1 (VR1) is a ligand-gated calcium channel that is believed to be the capsaicin-activated vanilloid receptor found in native tissues, based on similarities regarding molecular mass, tissue distribution, and electrophysiological properties. Using a Fluorescent Imaging Plate Reader (FLIPR), along with Fluo-3 to signal intracellular calcium levels ([Ca(++)](i)), rat VR1 (rVR1) and a human orthologue (hVR1) were pharmacologically characterized with various VR1 ligands. HEK-293 cells, stably expressing rVR1 or hVR1, exhibited dose-dependent increases in [Ca(++)](i) when challenged with capsaicin (EC(50)s congruent with 10 nM). Responses to capsaicin were blocked by the VR1 antagonist capsazepine and were dependent on VR1 expression. Potencies for 10 structurally diverse VR1 agonists revealed rVR1 potencies highly correlated to that of hVR1 (R(2) = 0.973). However, a subset of agonists (tinyatoxin, gingerol, and zingerone) was approximately 10-fold more potent for rVR1 compared to hVR1. Schild analysis for blockade of capsaicin-induced responses by capsazepine was consistent with competitive antagonism, whereas ruthenium red displayed noncompetitive antagonism. Compared to rVR1, hVR1 was more sensitive to blockade by both antagonists. For both rVR1 and hVR1, time-response waveforms elicited by resiniferatoxin increased more gradually compared to other agonists. Tinyatoxin also displayed slow responses with hVR1 but showed rapid responses with rVR1. Thus, FLIPR technology can be used to readily reveal differences between rVR1 and hVR1 pharmacology with respect to potencies, efficacies, and kinetics for several VR1 ligands.     

Interaction between protein kinase Cmu and the vanilloid receptor type 1

The capsaicin receptor VR1 is a polymodal nociceptor activated by multiple stimuli. It has been reported that protein kinase C plays a role in the sensitization of VR1. Protein kinase D/PKCmu is a member of the protein kinase D serine/threonine kinase family that exhibits structural, enzymological, and regulatory features distinct from those of the PKCs, with which they are related. As part of our effort to optimize conditions for evaluating VR1 pharmacology, we found that treatment of Chinese hamster ovary (CHO) cells heterologously expressing rat VR1 (CHO/rVR1) with butyrate enhanced rVR1 expression and activity. The expression of PKCmu and PKCbeta1, but not of other PKC isoforms, was also enhanced by butyrate treatment, suggesting the possibility that these two isoforms might contribute to the enhanced activity of rVR1. In support of this hypothesis, we found the following. 1) Overexpression of PKCmu enhanced the response of rVR1 to capsaicin and low pH, and expression of a dominant negative variant of PKCmu reduced the response of rVR1. 2) Reduction of endogenous PKCmu using antisense oligonucleotides decreased the response of exogenous rVR1 expressed in CHO cells as well as of endogenous rVR1 in dorsal root ganglion neurons. 3) PKCmu localized to the plasma membrane when overexpressed in CHO/rVR1 cells. 4) PKCmu directly bound to rVR1 expressed in CHO cells as well as to endogenous rVR1 in dorsal root ganglia or to an N-terminal fragment of rVR1, indicating a direct interaction between PKCmu and rVR1. 5) PKCmu directly phosphorylated rVR1 or a longer N-terminal fragment (amino acids 1-118) of rVR1 but not a shorter one (amino acids 1-99). 6) Mutation of S116A in rVR1 blocked both the phosphorylation of rVR1 by PKCmu and the enhancement by PKCmu of the rVR1 response to capsaicin. We conclude that PKCmu functions as a direct modulator of rVR1.     

4-(2-pyridyl)piperazine-1-carboxamides: potent vanilloid receptor 1 antagonists

A series of 4-(2-pyridyl)piperazine-1-carboxamide analogues based on the lead compound 1 was synthesized and evaluated for VR1 antagonist activity in capsaicin-induced (CAP) and pH (5.5)-induced (pH) FLIPR assays in a rat VR1-expressing HEK293 cell line. Potent VR1 antagonists were identified through SAR studies. From these studies, 18 was found to be very potent in the in vitro assay [IC(50)=4.8 nM (pH) and 35 nM (CAP)] and orally available in rat (F%=15.1).     

Hypertension-Linked Mutation of α-Adducin Increases CFTR Surface Expression and Activity in HEK and Cultured Rat Distal Convoluted Tubule Cells

The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) activity and localization are influenced by the cytoskeleton, in particular by actin and its polymerization state. In this study we investigated whether the expression of the hypertensive mutations of α-adducin (G460W-S586C in humans, F316Y in rats), an actin capping protein, led to a functional modification of CFTR activity and surface expression. The experiments were performed on HEK293 T cells cotransfected with CFTR and the human wild type (WT) or G460W mutated α-adducin. In whole-cell patch-clamp experiments, both the CFTR chloride current and the slope of current activation after forskolin addition were significantly higher in HEK cells overexpressing the G460W adducin. A higher plasma membrane density of active CFTR channels was confirmed by cell-attached patch-clamp experiments, both in HEK cells and in cultured primary DCT cells, isolated from MHS (Milan Hypertensive Strain, a Wistar rat (Rattus norvegicus) hypertensive model carrying the F316Y adducin mutation), compared to MNS (Milan Normotensive Strain) rats. Western blot experiments demonstrated an increase of the plasma membrane CFTR protein expression, with a modification of the channel glycosylation state, in the presence of the mutated adducin. A higher retention of CFTR protein in the plasma membrane was confirmed both by FRAP (Fluorescence Recovery After Photobleaching) and photoactivation experiments. The present data indicate that in HEK cells and in isolated DCT cells the presence of the G460W-S586C hypertensive variant of adducin increases CFTR channel activity, possibly by altering its membrane turnover and inducing a retention of the channel in the plasmamembrane. Since CFTR is known to modulate the activity of many others transport systems, the increased surface expression of the channel could have consequences on the whole network of transport in kidney cells.     

Synthesis and evaluation of pyridazinylpiperazines as vanilloid receptor 1 antagonists

A structurally biased chemical library of pyridazinylpiperazine analogs was prepared in an effort to improve the pharmaceutical and pharmacological profile of the lead compound N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide (BCTC). The library was evaluated for VR1 antagonist activity in capsaicin-induced (CAP) and pH5.5-induced (pH) FLIPR assays in a human VR1-expressing HEK293 cell line. The most potent VR1 antagonists were found to have IC(50) values in the range of 9-200nM with improved pharmaceutical and pharmacological profiles versus the lead BCTC. These compounds represent possible second-generation BCTC analogs.     

N-oleoyldopamine,a novel endogenous capsaicin-like lipid that produces hyperalgesia

N-Arachidonoyldopamine (NADA) was recently identified as an endogenous ligand for the vanilloid type 1 receptor (VR1). Further analysis of the bovine striatal extract from which NADA was isolated indicated the existence of substances corresponding in molecular mass to N-oleoyldopamine (OLDA), N-palmitoyldopamine (PALDA), and N-stearoyldopamine (STEARDA). Quadrupole time-of-flight mass spectrometric analysis of bovine striatal extracts revealed the existence of OLDA, PALDA, and STEARDA as endogenous compounds in the mammalian brain. PALDA and STEARDA failed to affect calcium influx in VR1-transfected human embryonic kidney (HEK) 293 cells or paw withdrawal latencies from a radiant heat source, and there was no evidence of spontaneous pain behavior. By contrast, OLDA induced calcium influx (EC(50) = 36 nm), reduced the latency of paw withdrawal from a radiant heat source in a dose-dependent manner (EC(50) = 0.72 microg), and produced nocifensive behavior. These effects were blocked by co-administration of the VR1 antagonist iodo-resiniferatoxin (10 nm for HEK cells and 1 microg/50 micro;l for pain behavior). These findings demonstrate the existence of an endogenous compound in the brain that is similar to capsaicin and NADA in its chemical structure and activity on VR1. Unlike NADA, OLDA was only a weak ligand for rat CB1 receptors; but like NADA, it was recognized by the anandamide membrane transporter while being a poor substrate for fatty-acid amide hydrolase. Analysis of the activity of six additional synthetic and potentially endogenous N-acyldopamine indicated the requirement of a long unsaturated fatty acid chain for an optimal functional interaction with VR1 receptors.     

Biochemical characterization of the vanilloid receptor 1 expressed in a dorsal root ganglia derived cell line.

The vanilloid receptor VR1 is an ion channel predominantly expressed by primary sensory neurons involved in nociception. Here we describe its biochemical properties and assess the subcellular localization, the glycosylation state and the quaternary structure of VR1 expressed in HEK293 cells and in the DRG-derived cell line F-11 (N18TG2 mouse neuroblastoma x rat dorsal root ganglia, hybridoma). VR1 was found to be glycosylated in both cell types. Of the five potential N-glycosylation sites, the predicted transient receptor potential channel-like transmembrane folding proposes N604 is localized extracellularly. We used site-directed mutagenesis to mutate the Asn at position 604 to Thr. This mutated VR1 was not glycosylated, confirming the extracellular location of N604 and its role as the exclusive site of glycosylation of the VR1 protein. VR1 occured in high molecular mass complexes as assessed by blue native PAGE. In the presence of limited amounts of SDS dimers, trimers and tetramers of VR1 were observed, consistent with the predicted tetrameric quaternary structure of the receptor. Cross-linking with dimethyladipimidate yielded almost exclusively dimers. Whereas VR1 localized both to the plasma membrane and to intracellular membranes in HEK293 cells, it localized predominantly to the plasma membrane in F-11 cells. Using confocal laserscanning microscopy, we observed an enrichment of anti-VR1 immunoreactivity in neurite-like structures of F-11 cells. In the light of conflicting literature data on biochemical characteristics of VR1, our data suggest that dorsal root ganglion-derived F-11 cells provide a powerful experimental system for the study of VR1 biochemistry.     

The vanilloid receptor (VR1)-mediated effects of anandamide are potently enhanced by the cAMP-dependent protein kinase

The endogenous cannabinoid receptor ligand, anandamide (AEA), is a full agonist of the vanilloid receptor type 1 (VR1) for capsaicin. Here, we demonstrate that the potency and efficacy of AEA at VR1 receptors can be significantly increased by the concomitant activation of protein kinase A (PKA). In human embryonic kidney (HEK) cells over-expressing human VR1, AEA induces a rise in cytosolic Ca(2+) concentration that is mediated by this receptor. The EC(50) for this effect was decreased five-fold in the presence of forskolin (FRSK, 1-5 microM) or the cAMP analogue, 8-Br-cAMP (10-100 microM). The effects of 8-Br-cAMP and FRSK were blocked by a selective PKA inhibitor. The FRSK (10 nM) also potently enhanced the sensory neurone- and VR1-mediated constriction by AEA of isolated guinea-pig bronchi, and this effect was abolished by a PKA inhibitor. In rat dorsal root ganglia slices, AEA-induced release of substance P, an effect mediated by VR1 activation, was enhanced three-fold by FRSK (10 nM). Thus, the ability of AEA to stimulate sensory VR1, with subsequent neuropeptide release, appears to be regulated by the state of activation of PKA. This observation supports the hypothesis that endogenous AEA might stimulate VR1 under certain pathophysiological conditions.     

A型流感病毒H5N1的M2离子通道稳定细胞株的建立

A型流感病毒H5N1的M2离子通道(H5M2)基因经优化后由人工合成,适合于哺乳动物细胞中表达.通过酶切克隆于pcDNA4质粒,并在HEK293细胞中建立稳定细胞株.Western blotting和免疫荧光证实H5M2在稳定细胞中只有在四环素诱导下才能表达,并经膜片钳证实在HEK293细胞中表达的H5M2具有H 通道活性,为M2离子通道功能的研究和M2离子通道阻断剂筛选方法的建立提供了参考.     

Arachidonyl dopamine as a ligand for the vanilloid receptor VR1 of the rat

The vanilloid receptor VR1 is a nonspecific Ca(2+) channel, expressed in sensory neurons in the peripheral nervous system and in various brain regions, which is believed to be an important molecular integrator of several chemical (acid, vanilloids) and physical stimuli (heat) that cause pain. Recently, several endogenous ligands for VR1 have been identified such as arachidonyl ethanolamide (anandamide) and the more potent arachidonyl dopamine (AA-DO). Here, we further characterize AA-DO as a ligand for rat VR1, heterologously expressed in CHO and HEK293 cells. AA-DO inhibited the binding of [3H]RTX to VR1 with a K(d) value of 5.49 +/- 0.68 microM and with positive cooperativity (p = 1.89 +/- 0.27), indicating that AA-DO was about 5-fold more potent than anandamide in this system. The K(d) (9.7 +/- 3.3 microM), and p values (1.54 +/- 0.04) for the binding of AA-DO to spinal cord membranes are in good correlation with the CHO-VR1 data. AA-DO stimulated 45Ca(2+) uptake on CHO-VR1 and HEK-VR1 cells with EC(50) values of 4.76 +/- 1.43 and 7.17 +/- 1.64 microM and Hill coefficients of 1.28 +/- 0.11 and 1.13 +/- 0.13, respectively, consistent with the binding measurements. In contrast to anandamide, AA-DO induced virtually the same level of 45Ca(2+) uptake as did capsaicin (90 +/- 6.6% in the CHO cells expressing VR1 and 89.3 +/- 9.4% in HEK293 cells expressing VR1). In a time dependent fashion following activation, AA-DO partially desensitized VR1 both in 45Ca(2+) uptake assays (IC(50) = 3.24 +/- 0.84 microM, inhibition is 68.5 +/- 6.85%) as well as in Ca(2+) imaging experiments (35.8 +/- 5.1% inhibition) using the CHO-VR1 system. The extent of desensitization was similar to that caused by capsaicin itself. We conclude that AA-DO is a full agonist for VR1 with a potency in the low micromolar range and is able to significantly desensitize the cells in a time and dose dependent manner.     

The genomic organization of the gene encoding the vanilloid receptor: evidence for multiple splice variants

Vanilloid receptor subtype-1 (VR1) is a nonselective cation channel that is expressed in sensory neurons and is activated by multiple noxious stimuli. Rat Vr1, stretch-inactivated channel (SIC), and vanilloid receptor 5' splice variant (VR.5'sv) have been hypothesized to be derived from a common VR gene. Characterization of the genomic structure encoding the 5' portion of rat Vr1 confirmed that VR.5'sv is derived from the VR gene; however, SIC seemed to be derived from two related but independent genes. We also deduced the genomic organization of the human gene VR1. Comparative studies of rat and human VR genes showed substantial conservation in genomic organization. The splice site flanking exon-intron 7 in rat and human VR1 diverged from the expected consensus sequence; this may help to explain the skipping of exon 7 within VR.5'sv and other VR splice variants.     

Modulation of the Kv1.3 Potassium Channel by Receptor Tyrosine Kinases

The voltage-dependent potassium channel, Kv1.3, is modulated by the epidermal growth factor receptor (EGFr) and the insulin receptor tyrosine kinases. When the EGFr and Kv1.3 are coexpressed in HEK 293 cells, acute treatment of the cells with EGF during a patch recording can suppress the Kv1.3 current within tens of minutes. This effect appears to be due to tyrosine phosphorylation of the channel, as it is blocked by treatment with the tyrosine kinase inhibitor erbstatin, or by mutation of the tyrosine at channel amino acid position 479 to phenylalanine. Previous work has shown that there is a large increase in the tyrosine phosphorylation of Kv1.3 when it is coexpressed with the EGFr. Pretreatment of EGFr and Kv1.3 cotransfected cells with EGF before patch recording also results in a decrease in peak Kv1.3 current. Furthermore, pretreatment of cotransfected cells with an antibody to the EGFr ligand binding domain (α-EGFr), which blocks receptor dimerization and tyrosine kinase activation, blocks the EGFr-mediated suppression of Kv1.3 current. Insulin treatment during patch recording also causes an inhibition of Kv1.3 current after tens of minutes, while pretreatment for 18 h produces almost total suppression of current. In addition to depressing peak Kv1.3 current, EGF treatment produces a speeding of C-type inactivation, while pretreatment with the α-EGFr slows C-type inactivation. In contrast, insulin does not influence C-type inactivation kinetics. Mutational analysis indicates that the EGF-induced modulation of the inactivation rate occurs by a mechanism different from that of the EGF-induced decrease in peak current. Thus, receptor tyrosine kinases differentially modulate the current magnitude and kinetics of a voltage-dependent potassium channel.     

Characterization of the Dimerization of Metabotropic Glutamate Receptors Using an N-Terminal Truncation of mGluR1α

The metabotropic glutamate receptor mGluR1alpha in membranes isolated both from rat brain and from cell lines transfected with cDNA coding for the receptor migrates as a disulphide-bonded dimer on sodium dodecyl sulphate-polyacrylamide gels. Dimerization of mGluR1alpha takes place in the endoplasmic reticulum because it is not prevented by exposing transfected human embryonic kidney (HEK) 293 cells to the drug brefeldin A, a drug that prevents egress of proteins from the endoplasmic reticulum. Dimerization was also not dependent on protein glycosylation as it was not prevented by treatment of the cells with tunicamycin. Using a mammalian expression vector containing the N-terminal domain of mGluR1alpha, truncated just before the first transmembrane domain (NT-mGluR1alpha), we show that the N-terminal domain is secreted as a soluble disulphide-bonded dimeric protein. In addition, the truncated N-terminal domain can form heterodimers with mGluR1alpha when both proteins are cotransfected into HEK 293 cells. However, mGluR1alpha and its splice variant mGluR1beta did not form heterodimers in doubly transfected HEK 293 cells. These results show that although the N-terminal domain of mGluR1alpha is sufficient for dimer formation, other domains in the molecule must regulate the process.     

Functional Expression of Rat Nav1.6 Voltage-Gated Sodium Channels in HEK293 Cells: Modulation by the Auxiliary β1 Subunit

The Na_v1.6 voltage-gated sodium channel α subunit isoform is abundantly expressed in the adult rat brain. To assess the functional modulation of Na_v1.6 channels by the auxiliary β1 subunit we expressed the rat Na_v1.6 sodium channel α subunit by stable transformation in HEK293 cells either alone or in combination with the rat β1 subunit and assessed the properties of the reconstituted channels by recording sodium currents using the whole-cell patch clamp technique. Coexpression with the β1 subunit accelerated the inactivation of sodium currents and shifted the voltage dependence of channel activation and steady-state fast inactivation by approximately 5–7 mV in the direction of depolarization. By contrast the β1 subunit had no effect on the stability of sodium currents following repeated depolarizations at high frequencies. Our results define modulatory effects of the β1 subunit on the properties of rat Na_v1.6-mediated sodium currents reconstituted in HEK293 cells that differ from effects measured previously in the Xenopus oocyte expression system. We also identify differences in the kinetic and gating properties of the rat Na_v1.6 channel expressed in the absence of the β1 subunit compared to the properties of the orthologous mouse and human channels expressed in this system.     

Characterization of Kir1.1 channels with the use of a radiolabeled derivative of tertiapin

Inward rectifier potassium channels (Kir) play critical roles in cell physiology. Despite representing the simplest tetrameric potassium channel structures, the pharmacology of this channel family remains largely undeveloped. In this respect, tertiapin (TPN), a 21 amino acid peptide isolated from bee venom, has been reported to inhibit Kir1.1 and Kir3.1/3.4 channels with high affinity by binding to the M1-M2 linker region of these channels. The features of the peptide-channel interaction have been explored electrophysiologically, and these studies have identified ways by which to alter the composition of the peptide without affecting its biological activity. In the present study, the TPN derivative, TPN-Y1/K12/Q13, has been synthesized and radiolabeled to high specific activity with (125)I. TPN-Y1/K12/Q13 and mono-iodo-TPN-Y1/K12/Q13 ([(127)I]TPN-Y1/K12/Q13) inhibit with high affinity rat but not human Kir1.1 channels stably expressed in HEK293 cells. [(125)I]TPN-Y1/K12/Q13 binds in a saturable, time-dependent, and reversible manner to HEK293 cells expressing rat Kir1.1, as well as to membranes derived from these cells, and the pharmacology of the binding reaction is consistent with peptide binding to Kir1.1 channels. Studies using chimeric channels indicate that the differences in TPN sensitivity between rat and human Kir1.1 channels are due to the presence of two nonconserved residues within the M1-M2 linker region. When these results are taken together, they demonstrate that [(125)I]TPN-Y1/K12/Q13 represents the first high specific activity radioligand for studying rat Kir1.1 channels and suggest its utility for identifying other Kir channel modulators.     

Agonist-induced internalization of the metabotropic glutamate receptor 1a is arrestin- and dynamin-dependent

At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 microM; 30 min) the HA-mGluR1a underwent internalization to endosomes. Further quantification of receptor internalization was provided by ELISA experiments which showed rapid agonist-induced internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a internalization is an arrestin- and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 microM; 30 min) HA-mGluR1a internalization. In addition, wild-type arrestin-2-green fluorescent protein (arrestin-2-GFP) or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced internalization of mGluR1a is an arrestin- and dynamin-dependent process.     

Calcium-activated K+ channels increase cell proliferation independent of K+ conductance

The intermediate-conductance calcium-activated potassium channel (IK1) promotes cell proliferation of numerous cell types including endothelial cells, T lymphocytes, and several cancer cell lines. The mechanism underlying IK1-mediated cell proliferation was examined in human embryonic kidney 293 (HEK293) cells expressing recombinant human IK1 (hIK1) channels. Inhibition of hIK1 with TRAM-34 reduced cell proliferation, while expression of hIK1 in HEK293 cells increased proliferation. When HEK293 cells were transfected with a mutant (GYG/AAA) hIK1 channel, which neither conducts K(+) ions nor promotes Ca(2+) entry, proliferation was increased relative to mock-transfected cells. Furthermore, when HEK293 cells were transfected with a trafficking mutant (L18A/L25A) hIK1 channel, proliferation was also increased relative to control cells. The lack of functional activity of hIK1 mutants at the cell membrane was confirmed by a combination of whole cell patch-clamp electrophysiology and fura-2 imaging to assess store-operated Ca(2+) entry and cell surface immunoprecipitation assays. Moreover, in cells expressing hIK1, inhibition of ERK1/2 and JNK kinases, but not of p38 MAP kinase, reduced cell proliferation. We conclude that functional K(+) efflux at the plasma membrane and the consequent hyperpolarization and enhanced Ca(2+) entry are not necessary for hIK1-induced HEK293 cell proliferation. Rather, our data suggest that hIK1-induced proliferation occurs by a direct interaction with ERK1/2 and JNK signaling pathways.     

Role of glycosylation in corin zymogen activation

The cardiac serine protease corin is the pro-atrial natriuretic peptide convertase. Corin is made as a zymogen, which is activated by proteolytic cleavage. Previous studies showed that recombinant human corin expressed in HEK 293 cells was biologically active, but activated corin fragments were not detectable, making it difficult to study corin activation. In this study, we showed that recombinant rat corin was activated in HEK 293 cells, murine HL-1 cardiomyocytes, and rat neonatal cardiomyocytes. In these cells, activated corin represented a small fraction of the total corin molecules. The activation of recombinant rat corin was inhibited by small molecule trypsin inhibitors but not inhibitors for matrix metalloproteinases or cysteine proteases, suggesting that a trypsin-like protease activated corin in these cells. Glycosidase digestion showed that rat and human corin proteins contained substantial N-glycans but little O-glycans. Treatment of HEK 293 cells expressing rat corin with tunicamycin prevented corin activation and inhibited its pro-atrial natriuretic peptide processing activity. Similar effects of tunicamycin on endogenous corin activity were found in HL-1 cells. Mutations altering the two N-glycosylation sites in the protease domain of rat corin prevented its activation in HEK 293 and HL-1 cells. Our results indicate that N-linked oligosaccharides play an important role in corin activation.     

Transient expression of vanilloid receptor subtype 1 in rat cardiomyocytes during development

Vanilloid receptor subtype 1 (VR1) is a non-selective cation channel detected on sensory neurons that is sensitive to capsaicin, the main pungent ingredient of hot chili pepper. Capsaicin application to neonatal animals causes cardiorespiratory distress, and this susceptibility to capsaicin changes during early postnatal life. This prompted us to hypothesise that in addition to its known neuronal localisation, VR1 is also expressed by non-neuronal cells of the heart during development. This issue was addressed in the rat heart during pre- and postnatal development by means of RT-PCR, western blot and immunohistochemistry. VR1-mRNA was transiently expressed from E14 to P30 but absent from adult hearts. Translation into protein was verified by western blotting. Immunohistochemistry proved that VR1 protein was localised in cardiomyocytes during those developmental stages at which mRNA was detected. In conclusion, VR1 is not neuron-specific but is transiently expressed on cardiomyocytes during ontogeny thereby pointing to a developmental role of this cation channel.