Memantine inhibits α3β2-nAChRs-mediated nitrergic neurogenic vasodilation in porcine basilar arteries

Abstract

Memantine, an NMDA receptor antagonist used for treatment of Alzheimer's disease (AD), is known to block the nicotinic acetylcholine receptors (nAChRs) in the central nervous system (CNS). In the present study, we examined by wire myography if memantine inhibited α3β2-nAChRs located on cerebral perivascular sympathetic nerve terminals originating in the superior cervical ganglion (SCG), thus, leading to inhibition of nicotine-induced nitrergic neurogenic dilation of isolated porcine basilar arteries. Memantine concentration-dependently blocked nicotine-induced neurogenic dilation of endothelium-denuded basilar arteries without affecting that induced by transmural nerve stimulation, sodium nitroprusside, or isoproterenol. Furthermore, memantine significantly inhibited nicotine-elicited inward currents in Xenopous oocytes expressing α3β2-, α7- or α4β2-nAChR, and nicotine-induced calcium influx in cultured rat SCG neurons. These results suggest that memantine is a non-specific antagonist for nAChR. By directly inhibiting α3β2-nAChRs located on the sympathetic nerve terminals, memantine blocks nicotine-induced neurogenic vasodilation of the porcine basilar arteries. This effect of memantine is expected to reduce the blood supply to the brain stem and possibly other brain regions, thus, decreasing its clinical efficacy in the treatment of Alzheimer's disease.

Figure 1. Effects of memantine on α3β2-nAChR-mediated nicotine-induced vasodilation in porcine basilar arteries.

All experiments were carried out in endothelium-denuded porcine basilar arteries in the presence of active muscle tone induced by U46619 (0.2 µM). A representative tracing showing that memantine (3 µM) blocked nicotine (100 µM)-induced vasodilation (panel A) without affecting that elicited by TNS (8 Hz). Summaries of memantine blockade of nicotine (100 µM)-induced vasodilation with IC₅₀ values are shown in panel B. Failure of memantine to inhibit relaxation elicited by TNS is summarized in panel C. Arrowheads in panel A indicate repeated washings (W). Vasodilation is estimated as percent of papaverine (PPV, 100 µM)-induced maximum vasodilation. Values are means ± SEM; n indicates number of experiments. *P<0.05 indicates significantly different from control.

Figure 2. Effects of memantine on isoproterenol (ISO)- and sodium nitroprusside (SNP)-induced vasorelaxation.

All experiments were carried out in endothelium-denuded porcine basilar arteries in the presence of active muscle tone induced by U46619 (0.2 µM). Panel A, a representative tracing showing that ISO (10⁻⁸ M–10⁻⁵ M) induced a concentration-dependent vasodilation which was not affected by memantine (10 µM). In panels B and C, concentration-response curves showing effects of memantine on ISO- and SNP-induced relaxation in porcine basilar arteries. The EC₅₀ values of ISO in inducing relaxation in the presence and absence of memantine were 0.06 (0.02–0.18) µM and 0.03 (0.01–0.09) µM, respectively. The EC₅₀ values of SNP in inducing relaxation in the presence and absence of memantine were 0.61 (0.2–2) µM and 0.71 (0.29–1.76) µM, respectively. The EC₅₀ values for isoproterenol and sodium nitroprusside-induced relaxation were not significantly different between the control and that in the presence of memantine (p>0.05). The values are mean ± SEM. n, number of experiments. Relaxation is estimated as percent of PPV (100 µM)-induced maximum relaxation.

Figure 3. Effects of memantine on nicotine-induced inward currents in xenopus oocytes expressing α7-nAChRs, α4β2-nAChRs, or α3β2-nAChRs.

A representative tracing in panel A from two-electrode voltage clamp studies shows that memantine (100 µM) blocks the inward currents elicited by nicotine (100 µM) in an oocyte expressing α4β2-nAChR. Oocytes were continuously perfused with memantine for 5 mins as indicated by the long horizontal open bar. Short horizontal solid bar above the tracing in panel A denotes application of nicotine for 3 sec. The blockade was completely reversed after washing off memantine. In panel B, memantine in a concentration-dependent manner blocked α7-, α4β2-, and α3β2-nAChRs-mediated inward currents elicited by nicotine (100 µM) with the IC₅₀ values as indicated. Arrows in panel A indicate repeated washings (W). Inward currents were estimated as a percentage of those induced by 100 µM nicotine. Values are means ± SEM; n indicates number of experiments.

Figure 4. Effects of memantine on nicotine-induced calcium influx in cultured rat superior cervical ganglion (SCG) neurons.

The neurons were loaded with fluo-4, AM (1 µM) in physiologic buffer and incubated at room temperature for 30 mins. Panel A showing the basal calcium image in each cell. In panel B, nicotine (100 µM) was applied to the medium to induce significant calcium influx into the neurons. Panel C showing that memantine (100 µM) significantly inhibited nicotine-induced calcium influx. In panel D, a complete recovery from the blockade of nicotine-induced calcium influx by memantine was observed 15 mins after washing off memantine. Values are means ± SEM; n indicates number of experiments. *P<0.05 indicates significantly different from control.

Figure 5. Summaries of effects of memantine on calcium influxes induced by nicotine (A) and KCl (B) in cultured neurons of the rat superior cervical ganglion (SCG).

The SCG neurons were loaded with fluo-4, AM (1 µM) in physiologic buffer and incubated at room temperature for 30 mins. Nicotine (100 µM) or KCl (50 mM) was then applied and the intracellular calcium measured as shown in Figure 4. Memantine in 3 µM to 300 µM was added 15 mins before application of nicotine (100 µM, panel A) or KCl (50 mM, panel B) to induce calcium influx, [Ca²⁺]_i. Each concentration of memantine was examined separately in each preparation. Memantine concentration-dependently inhibited nicotine (100 µM)-induced calcium influx (with IC₅₀ values shown in panel A). Effects of memantine on KCl (50 mM)-induced calcium influx are summarized and shown in panel B. Changes in intracellular calcium is estimated as percent of nicotine (100 µM)- or KCl (50 mM)-induced calcium influx. Values are means ± SEM; n indicates number of experiments. *P<0.05 indicates significantly different from the respective control.

Figure 6. Schematic illustration of the role of memantine in modulating parasympathetic nitrergic dilation of basilar arteries.

An axo-axonal interaction between adrenergic and nitrergic nerves in regulating vascular tone of the large cerebral arteries at the base of the porcine brain is shown. Nicotine activates presynaptic α3β2-nAChRs (in pigs crossbred among Landrace, Yorkshire and Duroc) or α7-nAChRs (in pigs crossbred between Landrace and Yorkshire) , on sympathetic nerves causing release of NE which then activates the presynaptic β2-adrenoceptors located on neighboring nitrergic nerves, causing NO release from nitrergic nerve terminals. NO activates guanylate cyclase (GC) in the smooth muscle cell to increase the synthesis of cGMP from GTP and relaxes the smooth muscle. NO is synthesized from L-arginine (L-Arg) in the presence of NO synthase (NOS). The byproduct of NO synthesis, L-citrulline (L-Cit), is actively converted to L-Arg . Memantine inhibits both presynaptic α7- and α3β2-nAChR-mediated nitrergic vasodilation.