pyrimidinones and capsazepine

Using human airway epithelial cell lines (i.e. NCI-H441 and Calu-3) as well as human alveolar epithelial type I-like (ATI) cells in primary culture, we studied the contribution of the epithelial sodium channel δ-subunit (δ-ENaC) to transepithelial sodium transport in human lung in vitro. Endogenous δ-ENaC protein was present in all three cell types tested; however, protein abundance was low, and no expression was detected in the apical cell membrane of these cells. Similarly, known modulators of δ-ENaC activity, such as capsazepine and icilin (activators) and Evans blue (inhibitor), did not show effects on short-circuit current (I SC), suggesting that δ-ENaC is not involved in the modulation of transcellular sodium absorption in NCI-H441 cell monolayers. Over-expression of δ-ENaC in NCI-H441 cells resulted in detectable protein expression in the apical cell membrane, as well as capsazepine and icilin-stimulated increases in I SC that were effectively blocked by Evans blue and that were consistent with δ-ENaC activation and inhibition, respectively. Consequently, these observations suggest that δ-ENaC expression is low in NCI-H441, Calu-3, and ATI cells and does not contribute to transepithelial sodium absorption.

Topics: Capsaicin; Diuretics; Epithelial Cells; Epithelial Sodium Channels; Evans Blue; Gene Knockdown Techniques; Humans; Primary Cell Culture; Pulmonary Alveoli; Pyrimidinones; Respiratory Mucosa; Sodium

Changes in the concentration of intracellular Ca(2+) ([Ca(2+) ]i) trigger and/or regulate principal sperm functions during fertilization, such as motility, capacitation, and the acrosome reaction (AR). Members of the large TRP channel family participate in a variety of Ca(2+) -dependent cell signaling processes. The eight TRPM channel members constitute one of the seven groups belonging to this family. Here we document using RT-PCR experiments the presence of Trpm2, 4, 7, and 8 in mouse spermatogenic cells. Trpm8 transcription is up-regulated after day 30. The localization of TRPM8 protein in mouse sperm was confirmed by immunocytochemistry and Western blots. Patch clamp recordings in testicular mouse sperm revealed TRPM8 agonist (menthol and icilin) activated currents sensitive to TRPM8 inhibitors N-(4-t-Butylphenyl)-4-(3-Chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide (BCTC) and capsazepine. These findings are consistent with the presence of functional TRPM8 in mouse sperm. Furthermore, menthol induced a [Ca(2+) ]i increase and the AR in these cells, that were inhibited by capsazepine (20 µM) and BCTC (1.6 µM). Notably, the progesterone and zona pellucida-induced AR was significantly (>40%) inhibited by BCTC and capsazepine, suggesting the possible participation of TRPM8 channels in this reaction. TRPM family members present in sperm could be involved in other important signaling events, such as thermotaxis, chemotaxis, and mechanosensory transduction.

Topics: Acrosome Reaction; Animals; Capsaicin; Humans; Ion Channel Gating; Male; Menthol; Mice; Mice, Knockout; Pyrazines; Pyridines; Pyrimidinones; Spermatozoa; Temperature; Testis; TRPM Cation Channels

The possible functional role of transient receptor potential (TRP) channels was investigated by testing various TRP agonists and antagonists in an isolated rat sinus hair follicle preparation. Extracellular recordings from slowly adapting type II mechanoreceptor units were made. The antagonist capsazepine depressed spontaneous and mechanically evoked activity, with an IC(50) of 82 microM. In one-third of units, capsazepine caused a selective depression of mechanically evoked firing, such that the existing spontaneous firing was interrupted by an absence of activity during the mechanical stimulus. The broad spectrum TRP blocker ruthenium red (30 microM) had inconsistent effects, although in some units a delayed onset (following wash) bursting and paroxysmal firing ensued. The agonist icilin (50-100 microM) had an excitatory effect on spontaneous firing, and (-)-menthol (200 microM) had inconsistent effects. Cinnamaldehyde (1-2 mM) depressed all types of activity equally, mechanically evoked and spontaneous. Camphor (0.5-2 mM) also depressed all types of activity, although it had a preferential effect on spontaneous activity. Capsaicin (1-10 microM) and allyl isothiocyanate (50-100 microM) had no clear effects. These results rule out any role for TRPA1 and TRPV1 channels in mechanotransduction processes of slowly adapting type II mechanoreceptors.

Topics: Acrolein; Action Potentials; Animals; Ankyrins; Calcium Channels; Camphor; Capsaicin; Evoked Potentials; Hair Follicle; In Vitro Techniques; Isothiocyanates; Male; Mechanoreceptors; Menthol; Physical Stimulation; Pyrimidinones; Rats; Rats, Wistar; Ruthenium Red; Time Factors; Transient Receptor Potential Channels; TRPA1 Cation Channel; TRPC Cation Channels; TRPV Cation Channels

The amiloride-sensitive epithelial Na(+) channel (ENaC) regulates Na(+) homeostasis in cells and across epithelia. Four homologous ENaC subunits (alpha, beta, gamma, and delta) have been isolated in mammals. The chemical activators acting on ENaC, however, are largely unknown. More recently, we have found that capsazepine activates human ENaCdelta (hENaCdelta), which is mainly expressed in the brain. In addition, here we show that icilin, which is a tetrahydropyrimidine-2-one derivative unrelated structurally to capsazepine, markedly enhanced the activity of hENaCdeltabetagamma heteromultimer expressed in Xenopus laevis oocytes. The inward currents at a holding potential of -60 mV in hENaCdeltabetagamma-expressing oocytes were increased by the application of icilin in a concentration-dependent manner with an EC(50) value of 33 microM. The icilin-elicited current was mostly abolished by the addition of 100 microM amiloride or by the removal of external Na(+). Homomeric hENaCdelta was also significantly activated by icilin, whereas hENaCalpha activity was not affected by icilin, and icilin caused a slight inhibition of the hENaCalphabetagamma current. Furthermore, icilin acted together with protons or capsazepine on hENaCdeltabetagamma. These findings identify icilin as a novel chemical activator of ENaCdelta, providing us with a lead compound for drug development in the degenerin/ENaC superfamily.

Topics: Amiloride; Animals; Capsaicin; Epithelial Sodium Channels; Humans; Protons; Pyrimidinones; Sodium Channel Agonists; Sodium Channels; Xenopus laevis