n-oleoyldopamine and resiniferatoxin

Colonic chloride secretion is regulated via the neurohormonal and immune systems. Exogenous chemicals (e.g., butyrate, propionate) can affect chloride secretion. Capsaicin, the pungent ingredient of the chili peppers, exerts various effects on gastrointestinal function. Capsaicin is known to activate the transient receptor potential vanilloid type 1 (TRPV1), expressed in the mesenteric nervous system. Recent studies have also demonstrated its presence in epithelial cells but its role remains uncertain. Because capsaicin has been reported to inhibit colonic chloride secretion, we tested whether this effect of capsaicin could occur by direct action on epithelial cells. In mouse colon and model T84 human colonic epithelial cells, we found that capsaicin inhibited forskolin-dependent short-circuit current (FSK-I(sc)). Using PCR and Western blot, we demonstrated the presence of TRPV1 in colonic epithelial cells. In T84 cells, TRPV1 localized at the basolateral membrane and in vesicular compartments. In permeabilized monolayers, capsaicin activated apical chloride conductance, had no effect on basolateral potassium conductance, but induced NKCC1 internalization demonstrated by immunocytochemistry and basolateral surface biotinylation. AMG-9810, a potent inhibitor of TRPV1, did not prevent the inhibition of the FSK-I(sc) by capsaicin. Neither resiniferatoxin nor N-oleoyldopamine, two selective agonists of TRPV1, blocked the FSK-I(sc). Conversely capsaicin, resiniferatoxin, and N-oleoyldopamine raised intracellular calcium ([Ca(2+)](i)) in T84 cells and AMG-9810 blocked the rise in [Ca(2+)](i) induced by capsaicin and resiniferatoxin suggesting the presence of a functional TRPV1 channel. We conclude that capsaicin inhibits chloride secretion in part by causing NKCC1 internalization, but by a mechanism that appears to be independent of TRPV1.

Topics: Acrylamides; Animals; Biotinylation; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Capsaicin; Cell Line; Chlorides; Colforsin; Colon; Diterpenes; Dopamine; Dose-Response Relationship, Drug; Electric Conductivity; Epithelial Cells; Humans; Immunohistochemistry; Intestinal Mucosa; Mice; Mice, Inbred C57BL; Reverse Transcriptase Polymerase Chain Reaction; Sodium-Potassium-Chloride Symporters; Solute Carrier Family 12, Member 2; Time Factors; TRPV Cation Channels

Agonists of the TRPV1 receptor excite TRPV1-expressing polymodal nociceptors that is followed after higher doses by a state of diminished responsiveness called desensitization which ensues at two levels: (i) diminished responsiveness of the ion channel (TRPV1 receptor desensitization); (ii) diminished responsiveness of the nerve endings to all stimuli including noxious heat. The aim was to compare these desensitizing actions of TRPV1 agonists in the rat by measuring with an incremental hot/cold plate the noxious heat and cold thresholds, i.e. the lowest hot and highest cold plate temperature, respectively, that evokes nocifensive behaviour. Capsaicin (3.3-1000 nmol) or resiniferatoxin (0.016-0.5 nmol) applied intraplantarly evoked a sustained dose-dependent elevation of the noxious heat threshold lasting for 2-11 days. N-oleoyldopamine failed to elevate the heat threshold. The noxious cold threshold was decreased by capsaicin or resiniferatoxin with a recovery within 2-4 days. The diminished acute nocifensive and heat threshold-lowering effects of resiniferatoxin or N-oleoyldopamine by pretreatment with doses that failed to elevate the heat threshold and to alter the nocifensive action of the TRPA1 activator formaldehyde, were taken as indication of TRPV1 receptor desensitization. In conclusion, using measurement of threshold temperatures eliciting nocifensive reactions in rats both in the hot and cold range revealed that capsaicin and RTX impair thermosensation in both noxious ranges due to a functional desensitization of peripheral terminals of TRPV1-expressing sensory neurons responsible for noxious heat and cold responsiveness. This could be differentiated from desensitization of TRPV1 receptor evoked by lower doses of resiniferatoxin or N-oleoyldopamine.

Topics: Analysis of Variance; Animals; Capsaicin; Cold Temperature; Diterpenes; Dopamine; Dose-Response Relationship, Drug; Female; Hot Temperature; Nociceptors; Pain Measurement; Pain Threshold; Rats; Rats, Wistar; Sensory System Agents; Thermosensing; TRPV Cation Channels

Transient receptor potential vanilloid receptor-1 (TRPV1) is a sensory neuron-specific cation channel capable of integrating various noxious chemical and physical stimuli. The dog orthologue of TRPV1 was cloned using cDNA from nodose ganglia and heterologously expressed in HEK293(OFF) cells. At the amino acid level, dTRPV1 displays 85-89% sequence identity to other TRPV1 orthologues. Molecular pharmacological characterization of HEK293(OFF) cells expressing TRPV1 was assessed using a fluorescence imaging plate reader (FLIPR)-based calcium imaging assay. Dog TRPV1 was activated by various known TRPV1 agonists in a concentration-dependent manner: Ag23 = resiniferatoxin > olvanil approximately arvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) > N-oleoyldopamine (OLDA). In addition, select TRPV1 antagonists (capsazepine, I-resiniferatoxin and N-(-4-tertiarybutylphenyl)-4-(3-cholorpyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC)) were able to block the response of dTRPV1 to capsaicin. Furthermore, the dog TRPV1 lacked a conserved protein kinase A (PKA) phosphorylation site (117) found in other cloned orthologues, which may have physiological consequences on dog TRPV1 function. Taken together, these data constitute the first study of the cloning, expression and pharmacological characterization of dog TRPV1.

Topics: Amino Acid Sequence; Animals; Biological Transport; Calcium; Capsaicin; Cell Line; Cloning, Molecular; Diterpenes; DNA, Complementary; Dogs; Dopamine; Dose-Response Relationship, Drug; Fluorometry; Genetic Vectors; Genotype; Humans; Molecular Sequence Data; Mutation, Missense; Phorbol Esters; Phylogeny; Pyrazines; Pyridines; Receptors, Drug; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Transfection