Signaling mechanisms of down-regulation of voltage-activated Ca²⁺ channels by transient receptor potential vanilloid type 1 stimulation with olvanil in primary sensory neurons

Olvanil ((N-vanillyl)-9-oleamide), a non-pungent transient receptor potential vanilloid type 1 agonist, desensitizes nociceptors and alleviates pain. But its molecular targets and signaling mechanisms are little known. Calcium influx through voltage-activated Ca²⁺ channels plays an important role in neurotransmitter release and synaptic transmission. Here we determined the effect of olvanil on voltageactivated Ca²⁺ channel currents and the signaling pathways in primary sensory neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated rat dorsal root ganglion neurons. Olvanil (1 μM) elicited a delayed but sustained inward current, and caused a profound inhibition (~60%) of N-, P/Q-, L-, and R-type voltage-activated Ca²⁺ channel current. Pretreatment with a specific transient receptor potential vanilloid type 1 antagonist or intracellular application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid abolished the inhibitory effect of olvanil on voltageactivated Ca²⁺ channel current. Calmodulin antagonists (ophiobolin-A and calmodulin inhibitory peptide) largely blocked the effect of olvanil and capsaicin on voltage-activated Ca²⁺ channel current. Furthermore, calcineurin (protein phosphatase 2B) inhibitors (deltamethrin and FK-506) eliminated the effect of olvanil on voltage-activated Ca²⁺ channel current. Notably, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin antagonists, and calcineurin inhibitors each alone significantly increased the amplitude of voltage-activated Ca²⁺ channel current. In addition, double immunofluorescence labeling revealed that olvanil induced a rapid internalization of Ca_V2.2 immunoreactivity from the membrane surface of dorsal root ganglion neurons. Collectively, this study suggests that stimulation of nonpungent transient receptor potential vanilloid type 1 inhibits voltage-activated Ca²⁺ channels through a biochemical pathway involving intracellular Ca²⁺-calmodulin and calcineurin in nociceptive neurons. This new information is important for our understanding of the signaling mechanisms of desensitization of nociceptors by transient receptor potential vanilloid type 1 analogues and the feedback regulation of intracellular Ca²⁺ and voltage-activated Ca²⁺ channels in nociceptive sensory neurons.