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Chem Biol Interact
2024 Aug 01;402:111213. doi: 10.1016/j.cbi.2024.111213.
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New insights into the effects of organometallic ruthenium complexes on nicotinic acetylcholine receptors.
Trobec T
,
Lamassiaude N
,
Benoit E
,
Žužek MC
,
Sepčić K
,
Kladnik J
,
Turel I
,
Aráoz R
,
Frangež R
.
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Nicotinic acetylcholine receptors (nAChRs) are expressed in excitable and non-excitable cells of the organism. Extensive studies suggest that nAChR ligands have therapeutic potential, notably for neurological and psychiatric disorders. Organometallic ruthenium complexes are known to inhibit several medically important enzymes such as cholinesterases. In addition, they can also interact with muscle- and neuronal-subtype nAChRs. The present study aimed to investigate the direct effects of three organometallic ruthenium complexes, [(η6-p-cymene)Ru(II)(5-nitro-1,10-phenanthroline)Cl]Cl (C1-Cl), [(η6-p-cymene)Ru(II)(1-hydroxypyridine-2(1H)-thionato)Cl] (C1a) and [(η6-p-cymene)Ru(II)(1-hydroxy-3-methoxypyridine-2(1H)-thionato)pta]PF6 (C1), on muscle-subtype (Torpedo) nAChRs and on the two most abundant human neuronal-subtype nAChRs in the CNS (α4β2 and α7) expressed in Xenopus laevis oocytes, using the two-electrode voltage-clamp. The results show that none of the three compounds had agonistic activity on any of the nAChR subtypes studied. In contrast, C1-Cl reversibly blocked Torpedo nAChR (half-reduction of ACh-evoked peak current amplitude by 332 nM of compound). When tested at 10 μM, C1-Cl was statistically more potent to inhibit TorpedonAChR than α4β2 and α7 nAChRs. Similar results of C1 effects were obtained on Torpedo and α4β2 nAChRs, while no action of the compound was detected on α7 nAChRs. Finally, the effects of C1a were statistically similar on the three nAChR subtypes but, in contrast to C1-Cl and C1, the inhibition was hardly reversible. These results, together with our previous studies on isolated mouse neuromuscular preparations, strongly suggest that C1-Cl is, among the three compounds studied, the only molecule that could be used as a potential myorelaxant drug.
Fig. 1. Chemical structures of the organometallic ruthenium complexes C1–Cl, C1a and C1.
Fig. 2. Effects of C1–Cl on ACh-induced current through Torpedo nAChRs. (A) Representative traces of ACh-induced inward current recorded from an oocyte microtransplanted with Torpedo nAChRs, in response to applications of (i) 50 μM ACh for 15 s (three times), (ii) 0.1 μM C1–Cl for 45 s, (iii) 0.1 μM C1–Cl together with 50 μM ACh for 15 s, and (iv) 50 μM ACh for 15 s (wash) (bars above recordings). The holding potential was −60 mV. (B) Concentration-response curves of the blocking effects of C1–Cl (Inhibition, filled circles) and their reversibility (Wash, open circles) on ACh-evoked current recorded from oocytes microtransplanted with Torpedo nAChRs. Currents were elicited by 15 s application of (i) 50 μM ACh (three times, control), (ii) C1–Cl (0.001–50 μM) together with 50 μM ACh (one time), and (iii) again 50 μM ACh (one time, wash). The peak current amplitude recorded in response to either a given C1–Cl concentration together with ACh or ACh alone (wash) was normalized to the mean one triggered by the first three-time applications of ACh alone (control), and plotted against the logarithm of C1–Cl concentration. The theoretical curve was calculated from typical sigmoid non-linear regression through data points (r2 = 0.933), according to Equation (1) (see Material and Methods) with IC50 and nH values of 332 nM and 1.08, respectively. Mean ± S.E.M. of 4–8 oocytes.
Fig. 3. Effects of C1–Cl on voltage-dependence of activation of ACh-induced current through Torpedo nAChRs. (A) Upper: Protocole used to access to the voltage-dependence of activation of ACh-induced current consisting in a series of 90-ms voltage pulses (from −120 to 60 mV, in 20 mV steps) applied from a holding potential of −60 mV to oocytes microtransplanted with Torpedo nAChRs, during the maximal current. Lower: Representative traces of current recorded during the above protocol and elicited by either 50 μM ACh alone (control, black trace) or together with 332 nM C1–Cl (red trace). (B) Voltage-dependence of activation. The values of peak current amplitude were normalized to those obtained at −60 mV under control conditions. Mean ± S.E.M. of 4 oocytes. (C) Percentage of current inhibition calculated at each test potential. *: P < 0.034 versus control.
Fig. 4. Effects of C1–Cl, C1 and C1a on ACh-induced currents through muscle-subtype (Torpedo) and human neuronal-subtype (α4β2 and α7) nAChRs. (A) Representative traces of ACh-induced inward currents recorded from oocytes expressing Torpedo, α4β2 and α7 nAChRs, in response to applications of 50–100 μM ACh for 15 s (Torpedo nAChRs), 10 s (α4β2 nAChRs) and 5 s (α7 nAChRs) (three times), 50–100 μM ACh together with 10 μM C1–Cl (a), C1 (b) and C1a (c) for 15 s (Torpedo nAChRs), 10 s (α4β2 nAChRs) and 5 s (α7 nAChRs), and 50–100 μM ACh for 15 s (Torpedo nAChRs), 10 s (α4β2 nAChRs) and 5 s (α7 nAChRs) (bars above recordings). The holding potential was −60 mV. (B,C) Histograms of the blocking effects (B) of 10 μM C1–Cl, C1 and C1a and their reversibility (C) on ACh-evoked currents through Torpedo, α4β2 and α7 nAChRs, recorded as detailed above. The peak current amplitude recorded in response to applications of ACh and a given compound (B) or ACh alone (C) was normalized to that triggered by the first three-time applications of ACh alone (control). Mean ± S.E.M. of 4–8 oocytes. *: P ≤ 0.048 versus control.
