α7 nicotinic acetylcholine receptor interaction with G proteins in breast cancer cell proliferation, motility, and calcium signaling.

Chronic smoking is a primary risk factor for breast cancer due to the presence of various toxins and carcinogens within tobacco products. Nicotine is the primary addictive component of tobacco products and has been shown to promote breast cancer cell proliferation and metastases. Nicotine activates nicotinic acetylcholine receptors (nAChRs) that are expressed in cancer cell lines. Here, we examine the role of the α7 nAChR in coupling to heterotrimeric G proteins within breast cancer MCF-7 cells. Pharmacological activation of the α7 nAChR using choline or nicotine was found to increase proliferation, motility, and calcium signaling in MCF-7 cells. This effect of α7 nAChR on cell proliferation was abolished by application of Gαi/o and Gαq protein blockers. Specifically, application of the Gαi/o inhibitor pertussis toxin was found to abolish choline-mediated cell proliferation and intracellular calcium transient response. These findings were corroborated by expression of a G protein binding dominant negative nAChR subunit (α7345-348A), which resulted in significantly attenuating calcium signaling and cellular proliferation in response to choline. Our study shows a new role for G protein signaling in the mechanism of α7 nAChR-associated breast cancer growth.

Effects of saturated long-chain N-acylethanolamines on voltage-dependent Ca2+ fluxes in rabbit T-tubule membranes.

The effects of saturated long-chain (C: 16-22) N-acylethanolamines and a series of saturated fatty acids with the same length of carbon chains were investigated on depolarization-induced (45)Ca(2+) fluxes mediated by voltage-dependent Ca(2+) channels in transverse tubule membrane vesicles from rabbit skeletal muscle. Vesicles were loaded with (45)Ca(2+) and membrane potentials were generated by establishing potassium gradients across the vesicle using the ionophore valinomycin. Arachidonoylethanolamide and docosaenoylethanolamide but not palmitoylethanolamide and stearoylethanolamide (all 10 microM) caused a significant inhibition of depolarization-induced (45)Ca(2+) fluxes and specific binding of [(3)H]Isradipine to transverse tubule membranes. On the other hand, saturated fatty acids including palmitic, stearic, arachidic, and docosanoic acids (all 10 microM) were ineffective in functional and radioligand binding experiments. Additional experiments using endocannabinoid metabolites suggested that whereas ethanolamine and arachidic acids were ineffective, arachidonoylethanolamide inhibited Ca(2+) effluxes and specific binding of [(3)H]Isradipine. Further studies indicated that only those fatty acids containing ethanolamine as a head group and having a chain length of more than 18 carbons were effective in inhibiting depolarization-induced Ca(2+) effluxes and specific binding of [(3)H]Isradipine. In conclusion, results indicate that depending on the chain length and the head group of fatty acid, N-acylethanolamines have differential effects on the function of voltage-dependent Ca(2+) channels and on the specific binding of [(3)H]Isradipine in skeletal muscle membranes.

Differential effects of endogenous and synthetic cannabinoids on voltage-dependent calcium fluxes in rabbit T-tubule membranes: comparison with fatty acids.

The effects of cannabinoid receptor ligands including 2-arachidonoylglycerol, R-methanandamide, Delta9-THC (Delta9-tetrahydrocannabinol), WIN 55,212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one], CP 55,940 ([1alpha,2beta-(R)-5alpha]-(-)-5-(1,1-dimethyl)-2-[5-hydroxy-2-(3-hydroxypropyl) cyclohexyl-phenol]) and a series of fatty acids on depolarization-induced Ca2+ effluxes mediated by voltage-dependent Ca2+ channels were investigated comparatively in transverse tubule membrane vesicles from rabbit skeletal muscle. Vesicles were loaded with 45Ca2+ and membrane potentials were generated by establishing potassium gradients across the vesicle using the ionophore valinomycin. Endocannabinoids, 2-arachidonoylglycerol and R-methanandamide (all 10 microM), inhibited depolarization-induced Ca2+ effluxes and specific binding of [3H]PN 200-110 (isradipine) to transverse tubule membranes. On the other hand, synthetic cannabinoids, including CP 55,940, WIN 55,212-2, and Delta9-THC (all 10 microM), were ineffective. Additional experiments using endocannabinoid metabolites suggested that whereas ethanolamine and glycerol were ineffective, arachidonic acid inhibited Ca2+ effluxes and specific binding of [3H]PN 200-110. Further studies indicated that only those fatty acids containing two or more double bonds were effective in inhibiting depolarization-induced Ca2+ effluxes and specific binding of [3H]PN 200-110. These results indicate that endocannabinoids, but not synthetic cannabinoids, directly inhibit the function of voltage-dependent calcium channels (VDCCs) and modulate the specific binding of calcium channel ligands of the dihydropyridine (DHP) class.

Inhibition of cromakalim-activated K+ current by ethanol in follicle-enclosed Xenopus oocytes.

Ethanol has been reported to modulate arterial dilation and insulin secretion. ATP-inhibited K+ channels (K(ATP)) are reported to have regulatory roles during these events. In the present study, the effect of ethanol on K+ currents activated by the K(ATP) channel opener cromakalim was investigated in follicular cells of Xenopus oocytes. The results indicate that ethanol in the concentration range of 10-300 mM (approximately 0.06%-2% v/v) reversibly inhibits the cromakalim-induced K+ currents. The 50% of maximal ethanol effect was obtained at 53 mM. Inhibition of cromakalim-activated K+ current by ethanol was non-competitive. In oocytes treated with the Ca2+-chelator BAPTA, inhibition of cromakalim-induced K+ currents by ethanol was not altered, suggesting that Ca2+-activated second messenger pathways were not involved in the actions of ethanol. Similarly, currents activated by 8-Br-cAMP were also inhibited by ethanol, but the ethanol inhibition of 8-Br-cAMP-activated currents was significantly less than inhibition of cromakalim-activated currents by ethanol. These results indicate that cromakalim-activated K+ currents in follicular cells of Xenopus oocytes were modulated by ethanol.

Comparison of the effects of xenon and halothane on voltage-dependent Ca(2+) fluxes in rabbit T-tubule membranes.

The effects of xenon and halothane on depolarization-induced (45)Ca(2+) fluxes mediated by voltage-dependent Ca(2+) channels were investigated in transverse tubule membrane vesicles from rabbit skeletal muscle. Halothane, in the concentration range of 0.5-2 mM, caused a significant inhibition of (45)Ca(2+) fluxes. Xenon tested in the range of 60%-100% did not affect the (45)Ca(2+) fluxes. Radioligand binding studies indicated that xenon and halothane have different effects on the specific binding of [(3)H]Isradipine to transverse tubule membranes. Halothane caused a significant inhibition on the specific binding of [(3)H]Isradipine. In controls and in presence of 0.5 mM halothane, B(max) values were 26.9 pmole/mg and 15.1 pmole/mg, and K(D) values were 238 pM and 247 pM, respectively. On the other hand, there was no effect of xenon (60%-100%) on the characteristics of [(3)H]Isradipine binding. In conclusion, results indicate that xenon and halothane differ in their effects on the function of voltage-dependent Ca(2+) channels and on the specific binding of [(3)H]Isradipine in skeletal muscle membranes.

Effects of isoflurane on voltage-dependent calcium fluxes in rabbit T-tubule membranes: comparison with alcohols.

The effects of racemic (+/-) and (+)- and (-)-stereoisomers of isoflurane on depolarization-induced (45)Ca(2+) fluxes mediated by voltage-dependent Ca(2+) channels were investigated in transverse tubule membrane vesicles from rabbit skeletal muscle. In the concentration range 0.5 to 2 mM, (+/-)-isoflurane inhibited (45)Ca(2+) fluxes and functionally modulated the effects of the Ca(2+) channel antagonist nifedipine (1-10 microM). Isoflurane-induced inhibition of (45)Ca(2+) fluxes was not significantly affected by pretreatment with either pertussis toxin (5 microg/ml) or phorbol 12-myristate 13-acetate (50 nM). Further experiments indicated that there were no significant differences between (+)- and (-)-stereoisomers of isoflurane with respect to the extent of inhibition of (45)Ca(2+) fluxes. Radioligand binding studies indicated that racemic and (+)- and (-)-isoflurane were equally effective in displacing the specific binding of [(3)H]PN 200-110 to transverse tubule membranes. There were no apparent differences between the effects of (+)- and (-)-isoflurane on the characteristics of [(3)H]PN 200-110 binding. Although the concentrations of isoflurane for the inhibitions of (45)Ca(2+) fluxes and radioligand bindings were similar, the concentrations of n-alcohols required for the inhibition of (45)Ca(2+) fluxes were lower than those for the displacement of radioligand. Comparison of the data for the displacement of [(3)H]PN 200-110 binding and the inhibition of (45)Ca(2+) fluxes by isoflurane and by n-alcohols suggested that both isoflurane and n-alcohols may have more than a single binding site. In conclusion, results indicate that isoflurane, independent of intracellular Ca(2+) levels, nonstereospecifically inhibits the function of voltage-dependent Ca(2+) channels and this effect is mediated through multiple binding sites.