Activation of recombinant human TRPV1 receptors expressed in SH-SY5Y human neuroblastoma cells increases [Ca(2+)](i), initiates neurotransmitter release and promotes delayed cell death.
The transient receptor potential (TRP) vanilloid receptor subtype 1 (TRPV1) is a ligand-gated, Ca(2+)-permeable ion channel in the TRP superfamily of channels. We report the establishment of the first neuronal model expressing recombinant human TRPV1 (SH-SY5Y(hTRPV1)). SH-SY5Y human neuroblastoma cells were stably transfected with hTRPV1 using the Amaxa Biosystem (hTRPV1 in pIREShyg2 with hygromycin selection). Capsaicin, olvanil, resiniferatoxin and the endocannabinoid anandamide increased [Ca(2+)](i) with potency (EC(50)) values of 2.9 nmol/L, 34.7 nmol/L, 0.9 nmol/L and 4.6 micromol/L, respectively. The putative endovanilloid N-arachidonoyl-dopamine increased [Ca(2+)](i) but this response did not reach a maximum. Capsaicin, anandamide, resiniferatoxin and olvanil mediated increases in [Ca(2+)](i) were inhibited by the TRPV1 antagonists capsazepine and iodo-resiniferatoxin with potencies (K(B)) of approximately 70 nmol/L and 2 nmol/L, respectively. Capsaicin stimulated the release of pre-labelled [(3)H]noradrenaline from monolayers of SH-SY5Y(hTRPV1) cells with an EC(50) of 0.6 nmol/L indicating amplification between [Ca(2+)](i) and release. In a perfusion system, we simultaneously measured [(3)H]noradrenaline release and [Ca(2+)](i) and observed that increased [Ca(2+)](i) preceded transmitter release. Capsaicin treatment also produced a cytotoxic response (EC(50) 155 nmol/L) that was antagonist-sensitive and mirrored the [Ca(2+)](I) response. This model displays pharmacology consistent with TRPV1 heterologously expressed in standard non-neuronal cells and native neuronal cultures. The advantage of SH-SY5Y(hTRPV1) is the ability of hTRPV1 to couple to neuronal biochemical machinery and produce large quantities of cells.
Lam
Patricia M W
PM
Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group), Division of Anaesthesia, Critical Care and Pain Management, University of Leicester, Leicester Royal Infirmary, Leicester, UK.
Hainsworth
Atticus H
AH
Smith
Graham D
GD
Owen
Davina E
DE
Davies
James
J
Lambert
David G
DG
D001095:Arachidonic Acids / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics*; D002211:Capsaicin / Q000031:analogs & derivatives / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004224:Diterpenes / Q000494:pharmacology; D004298:Dopamine / Q000031:analogs & derivatives / Q000494:pharmacology; D063388:Endocannabinoids; D006801:Humans; D008954:Models, Biological; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism*; D053284:Polyunsaturated Alkamides / Q000494:pharmacology; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009435:Synaptic Transmission / Q000187:drug effects / Q000235:genetics; D050916:TRPV Cation Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
D001095:Arachidonic Acids; D004224:Diterpenes; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; D011994:Recombinant Proteins; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; C474941:arachidonyl dopamine; C468130:iodoresiniferatoxin; C054256:olvanil; C024353:resiniferatoxin; C071423:capsazepine; D002211:Capsaicin; D002118:Calcium; C078814:anandamide; D004298:Dopamine; D009638:Norepinephrine