Endogenous cannabinoid anandamide inhibits nicotinic acetylcholine receptor function in mouse thalamic synaptosomes.

The effects of the endogenous cannabinoid anandamide [arachidonylethanolamide (AEA)] on the function of nicotinic acetylcholine receptor (nAChR) were investigated using the 86Rb+ efflux assay in thalamic synaptosomes. AEA reversibly inhibited 86Rb+ efflux induced by 300 microM ACh with an IC50 value of 0.9 +/- 2 microM. Pre-treatment with the cannabinoid (CB1) receptor antagonist SR141716A (1 microM), the CB2 receptor antagonist SR144528 (1 microM), or pertussis toxin (0.2 mg/mL) did not alter the inhibitory effects of AEA, suggesting that known CB receptors are not involved in AEA inhibition of nAChRs. AEA inhibition of 86Rb+ efflux was not reversed by increasing acetylcholine (ACh) concentrations. In radioligand binding studies, the specific binding of [3H]-nicotine was not altered in the presence of AEA, indicating that AEA inhibits the function of nAChR in a non-competitive manner. Neither the amidohydrolase inhibitor phenylmethylsulfonyl fluoride (0.2 mM) nor the cyclooxygenase inhibitor, indomethacin, (5 microM) affected AEA inhibition of nAChRs, suggesting that the effect of AEA is not mediated by its metabolic products. Importantly, the extent of AEA inhibition of 86Rb+ efflux was significantly attenuated by the absence of 1% fatty acid free bovine serum albumin pre-treatment, supporting previous findings that fatty acid-like compounds modulate the activity of nAChRs. Collectively, the results indicate that AEA inhibits the function of nAChRs in thalamic synaptosomes via a CB-independent mechanism and that the background activity of these receptors is affected by fatty acids and AEA.

Immortalization and characterization of a nociceptive dorsal root ganglion sensory neuronal line.

Development of neuroprotective strategies for peripheral neuropathies requires high-throughput drug screening assays with appropriate cell types. Currently, immortalized dorsal root ganglion (DRG) sensory neuronal cell lines that maintain nociceptive sensory neuronal properties are not available. We generated immortalized DRG neuronal lines from embryonic day 14.5 rats. Here, we show that one of the immortalized DRG neuronal lines, 50B11, has the properties of a nociceptive neuron. When differentiated in the presence of forskolin, these cells extend long neurites, express neuronal markers, and generate action potentials. They express receptors and markers of small-diameter sensory neurons and upregulate appropriate receptor populations when grown in the presence of glial cell line-derived neurotrophic factor or nerve growth factor. Furthermore, they express capsaicin receptor transient receptor potential vanilloid family-1 (TRPV-1) and respond to capsaicin with increases in intracellular calcium. In a 96-well plate format, these neurons show a decline in ATP levels when exposed to dideoxycytosine (ddC) in a proper time- and dose-dependent manner. This ddC-induced reduction in ATP levels correlates with axonal degeneration. The immortalized DRG neuronal cell line 50B11 can be used for high-throughput drug screening for neuroprotective agents for axonal degeneration and antinociceptive drugs that block TRPV-1.

The solubilizing detergents, Tween 80 and Triton X-100 non-competitively inhibit alpha 7-nicotinic acetylcholine receptor function in Xenopus oocytes.

Because many studies rely upon detergents to solubilize lipophilic agents such as cannabinoid drugs, we examined the effect of commonly employed detergents on the function of the cloned alpha(7) subunit of the nicotinic ACh receptor. Homomeric alpha(7) receptors were expressed in Xenopus oocytes and the two-microelectrode voltage-clamp technique was used to assess their electrophysiological properties. The detergents Tween 80 and Triton X-100 reversibly inhibited ACh (100 microM)-induced inward currents in a concentration-dependent manner, with IC(50) values of 610 nM and 1.4 microM, respectively. The effects of these detergents were independent of membrane potential, and they were not mediated by endogenous Ca(2+)-dependent Cl(-) channels, since they were unaffected by intracellularly injected BAPTA, and recorded in Ca(2+)-free bathing solution containing 2 mM Ba(2+). Both detergents also decreased the maximal effect of ACh, without significantly affecting its EC(50), indicating a non-competitive interaction with the nACh alpha(7) receptors. In contrast to the effects of these detergents, we found that cholic acid (10 microM), DMSO (10 microM) and Tocrisol (0.01% v/v) did not cause a significant effect on nicotinic responses. In conclusion, we demonstrate that the detergents Tween 80 and Triton X-100 are potent inhibitors of neuronal nACh alpha(7) receptors expressed in Xenopus oocytes, and we suggest that studies utilizing these detergents to solubilize lipophilic drugs should be scrutinized for such effects.

Endogenous cannabinoid, anandamide, acts as a noncompetitive inhibitor on 5-HT3 receptor-mediated responses in Xenopus oocytes.

The cloned 5-HT3 receptor from NCB-20 neuroblastoma cells was expressed in Xenopus oocytes and the effect of the endogenous cannabinoid ligand, anandamide, was investigated on the function of this receptor. The oocytes expressing the cloned 5-HT3 receptors were voltage-clamped at -70 mV. Anandamide, at the concentration range of 0.1-100 microM, reversibly inhibited 1 microM 5-HT induced currents. The inhibition of 5-HT induced currents by anandamide was concentration-dependent with an EC50 of 3.7 microM and slope value of 0.94. This inhibitory effect was not dependent on the membrane potential and anandamide did not have an effect on the reversal potential of 5-HT-induced currents. In the presence of 10 microM anandamide, the maximum 5-HT-induced response was also inhibited and the respective EC50 values were 3.4 microM and 3.1 microM in the absence and presence of anandamide, indicating that anandamide acts as a noncompetitive antagonist on 5-HT3 receptors. CB1 receptor antagonist SR-141716A (1 microM) and pertussis toxin (5 microg/ml) did not cause a significant change on the inhibition of 5-HT responses by anandamide. The effect of anandamide was not changed by preincubating the oocytes with 0.2 mM 8-Br-cAMP, a membrane-permeable analog of cAMP, or Sp-cAMPS (0.1 mM), a membrane-permeable protein kinase A activator. These results suggest that the effect of anandamide is independent of the activation of cAMP pathway and not mediated by the activation of PTX sensitive G-proteins. In conclusion, we demonstrated that the endogenous cannabinoid anandamide inhibits the function of 5-HT3 receptors expressed in Xenopus oocytes in a cannabinoid-receptor independent and noncompetitive manner.