thiourea and anandamide

Odontoblasts are involved in the transduction of stimuli applied to exposed dentin. Although expression of thermo/mechano/osmo-sensitive transient receptor potential (TRP) channels has been demonstrated, the properties of TRP vanilloid 1 (TRPV1)-mediated signaling remain to be clarified. We investigated physiological and pharmacological properties of TRPV1 and its functional coupling with cannabinoid (CB) receptors and Na(+)-Ca(2+) exchangers (NCXs) in odontoblasts. Anandamide (AEA), capsaicin (CAP), resiniferatoxin (RF) or low-pH evoked Ca(2+) influx. This influx was inhibited by capsazepine (CPZ). Delay in time-to-activation of TRPV1 channels was observed between application of AEA or CAP and increase in [Ca(2+)](i). In the absence of extracellular Ca(2+), however, an immediate increase in [Ca(2+)](i) was observed on administration of extracellular Ca(2+), followed by activation of TRPV1 channels. Intracellular application of CAP elicited inward current via opening of TRPV1 channels faster than extracellular application. With extracellular RF application, no time delay was observed in either increase in [Ca(2+)](i) or inward current, indicating that agonist binding sites are located on both extra- and intracellular domains. KB-R7943, an NCX inhibitor, yielded an increase in the decay time constant during TRPV1-mediated Ca(2+) entry. Increase in [Ca(2+)](i) by CB receptor agonist, 2-arachidonylglycerol, was inhibited by CB1 receptor antagonist or CPZ, as well as by adenylyl cyclase inhibitor. These results showed that TRPV1-mediated Ca(2+) entry functionally couples with CB1 receptor activation via cAMP signaling. Increased [Ca(2+)](i) by TRPV1 activation was extruded by NCXs. Taken together, this suggests that cAMP-mediated CB1-TRPV1 crosstalk and TRPV1-NCX coupling play an important role in driving cellular functions following transduction of external stimuli to odontoblasts.

Topics: Animals; Arachidonic Acids; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Signaling; Cannabinoid Receptor Antagonists; Capsaicin; Cyclic AMP; Diterpenes; Endocannabinoids; Glycerides; Hydrogen-Ion Concentration; Odontoblasts; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptors, Cannabinoid; Sodium-Calcium Exchanger; Thiourea; TRPV Cation Channels

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-selective ion channel expressed in human lung cells. We show that activation of the intracellular subpopulation of TRPV1 causes endoplasmic reticulum (ER) stress and cell death in human bronchial epithelial and alveolar cells. TRPV1 agonist (nonivamide) treatment caused calcium release from the ER and altered the transcription of growth arrest- and DNA damage-inducible transcript 3 (GADD153), GADD45alpha, GRP78/BiP, ATF3, CCND1, and CCNG2) in a manner comparable with prototypical ER stress-inducing agents. The TRPV1 antagonist N-(4-tert-butylbenzyl)-N'-(1-[3-fluoro-4-(methylsulfonylamino)-phenyl]ethyl)thiourea (LJO-328) inhibited mRNA responses and cytotoxicity. EGTA and ruthenium red inhibited cell surface TRPV1 activity, but they did not prevent ER stress gene responses or cytotoxicity. Cytotoxicity paralleled eukaryotic translation initiation factor 2, subunit 1 (EIF2alpha) phosphorylation and the induction of GADD153 mRNA and protein. Transient overexpression of GADD153 caused cell death independent of agonist treatment, and cells selected for stable overexpression of a GADD153 dominant-negative mutant exhibited reduced sensitivity. Salubrinal, an inhibitor of ER stress-induced cytotoxicity via the EIF2alphaK3/EIF2alpha pathway, or stable overexpression of the EIF2alpha-S52A dominant-negative mutant also inhibited cell death. Treatment of the TRPV1-null human embryonic kidney 293 cell line with TRPV1 agonists did not initiate ER stress responses. Likewise, n-benzylnonanamide, an inactive analog of nonivamide, failed to cause ER calcium release, an increase in GADD153 expression, and cytotoxicity. We conclude that activation of ER-bound TRPV1 and stimulation of GADD153 expression via the EIF2alphaK3/EIF2alpha pathway represents a common mechanism for cytotoxicity by cell-permeable TRPV1 agonists. These findings are significant within the context of lung inflammatory diseases where elevated concentrations of endogenous TRPV1 agonists are probably produced in sufficient quantities to cause TRPV1 activation and lung cell death.

Topics: Activating Transcription Factor 3; Arachidonic Acids; Calcium; Capsaicin; Cell Line; Cell Line, Tumor; Cell Survival; Cells, Cultured; Cinnamates; Cyclin D1; Cyclin G2; Cyclins; Diterpenes; Dithiothreitol; Endocannabinoids; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Epithelial Cells; Eukaryotic Initiation Factor-2; Gene Expression; Humans; Lung; Phosphorylation; Polyunsaturated Alkamides; Thapsigargin; Thiourea; Transcription Factor CHOP; Transfection; TRPV Cation Channels

At birth, the increase in oxygen causes contraction of the ductus arteriosus, thus diverting blood flow to the lungs. Although this contraction is modulated by substances such as endothelin and dilator prostaglandins, normoxic contraction is an intrinsic property of ductus smooth muscle. Normoxic inhibition of potassium channels causes membrane depolarization and calcium entry through L-type calcium channels. However, the studies reported here show that after inhibition of this pathway there is still substantial normoxic contraction, indicating the involvement of additional mechanisms.. Using ductus ring experiments, calcium imaging, reverse-transcription polymerase chain reaction, Western blot, and cellular electrophysiology, we find that this depolarization-independent contraction is caused by release of calcium from the IP3-sensitive store in the sarcoplasmic reticulum, by subsequent calcium entry through store-operated channels, and by increased calcium sensitization of actin-myosin filaments, involving Rho-kinase.. Much of the normoxic contraction of the ductus arteriosus at birth is related to calcium entry through store-operated channels, encoded by the transient receptor potential superfamily of genes, and to increased calcium sensitization. A clearer understanding of the mechanisms involved in normoxic contraction of the ductus will permit the development of better therapy to close the patent ductus arteriosus, which constitutes approximately 10% of all congenital heart disease and is especially common in premature infants.

Topics: Animals; Arachidonic Acids; Boron Compounds; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium Signaling; Cytosol; Ductus Arteriosus; Endocannabinoids; Imidazoles; In Vitro Techniques; Indoles; Intracellular Signaling Peptides and Proteins; Isoquinolines; Maleimides; Menthol; Mibefradil; Muscle Contraction; Nifedipine; Niflumic Acid; Oxidation-Reduction; Oxygen; Patch-Clamp Techniques; Polyunsaturated Alkamides; Potassium Channels; Protein Serine-Threonine Kinases; Rabbits; rho-Associated Kinases; Ruthenium Red; Sulfonamides; Tetraethylammonium; Thapsigargin; Thiourea