In vitro and in silico analysis of the vascular effects of asymmetrical N,N-bis(alkanol)amine aryl esters, novel multidrug resistance-reverting agents.

Asymmetrical N,N-bis(alkanol)amine aryl esters (FRA77, GDE6, and GDE19) are potent multidrug resistance (MDR) reversers. Their structures loosely remind that of the Ca(2+) antagonist verapamil. Therefore, the aim of this study was to investigate their vascular activity in vitro. Their effects on the mechanical activity of fresh and cultured rat aorta rings on Cav1.2 channel current (I Ca1.2) of A7r5 cells and their cytotoxicity on A7r5 and EA.hy926 cells were analyzed. Docking at the rat α1C subunit of the Cav1.2 channel was simulated in silico. Compounds tested were cytotoxic at concentrations >1 µM (FRA77, GDE6, GDE19) and >10 µM (verapamil) in EA.hy926 cells, or >10 µM (FRA77, GDE6, GDE19) and at 100 µM (verapamil) in A7r5 cells. In fresh rings, the three compounds partly antagonized phenylephrine and 60 mM K(+) (K60)-induced contraction at concentrations ≥1 and ≥3 µM, respectively. On the contrary, verapamil fully relaxed rings pre-contracted with both agents. In cultured rings, 10 µM GDE6, GDE19, FRA77, and verapamil significantly reduced the contractile response to both phenylephrine and K60. Similarly to verapamil, the three compounds docked at the α1C subunit, interacting with the same amino acids residues. FRA77, GDE6, and GDE19 inhibited I Ca1.2 with IC50 values 1 order of magnitude higher than that of verapamil. FRA77-, GDE6-, and GDE19-induced vascular effects occurred at concentrations that are at least 1 order of magnitude higher than those effectively reverting MDR. Though an unambiguous divergence between MDR reverting and vascular activity is of overwhelming importance, these findings consistently contribute to the design and synthesis of novel and potent chemosensitizers.

A transport mechanism for NAADP in a rat basophilic cell line.

NAADP is a second messenger that releases Ca2+ from intracellular stores. Surprisingly, it has been recently shown that extracellular application of NAADP is capable of inducing intracellular Ca2+ release. This is particularly important since the only mammalian enzymes known to catalyze the synthesis of this second messenger are located extracellularly. In the present manuscript, we have investigated whether mammalian cells possess a transport system capable of transporting this highly charged molecule into cells. Indeed, in RBL-2H3 cells, a rat basophilic cell line, and in SK-N-BE cells, a neuroblastoma cell line, [32P]NAADP is efficiently transported inside cells. NAADP transport is Na+ and Ca2+ dependent, is partially blocked by dipyridamole, but is unaffected by nitrobenzylthioinosine. RBL-2H3 cells also transport [32P]cADPR, but the differences in the pharmacological profile suggest that NAADP transport proceeds by a novel mechanism. Lastly, extracellular application of NAADP, but not NADP, induced a raise in intracellular Ca2+. This is the first demonstration that NAADP is transported into cells and highlights the possibility that, alongside a second messenger, NAADP might also act as an autocrine/paracrine signal.

Sphingosine releases Ca2+ from intracellular stores via the ryanodine receptor in sea urchin egg homogenates.

Various reports have demonstrated that the sphingolipids sphingosine and sphingosine-1-phosphate are able to induce Ca2+ release from intracellular stores in a similar way to second messengers. Here, we have used the sea urchin egg homogenate, a model system for the study of intracellular Ca2+ release mechanisms, to investigate the effect of these sphingolipids. While ceramide and sphingosine-1-phosphate did not display the ability to release Ca2+, sphingosine stimulated transient Ca2+ release from thapsigargin-sensitive intracellular stores. This release was inhibited by ryanodine receptor blockers (high concentrations of ryanodine, Mg2+, and procaine) but not by pre-treatment of homogenates with cADPR, 8-bromo-cADPR or blockers of other intracellular Ca2+ channels. However, sphingosine rendered the ryanodine receptor refractory to cADPR. We propose that, in the sea urchin egg, sphingosine is able to activate the ryanodine receptor via a mechanism distinct from that used by cADPR.