Vesna A. Eterović

Actions of octocoral and tobacco cembranoids on nicotinic receptors.

Nicotinic acetylcholine receptors (AChRs) are pentameric proteins that form agonist-gated cation channels through the plasma membrane. AChR agonists and antagonists are potential candidates for the treatment of neurodegenerative diseases. Cembranoids are naturally occurring diterpenoids that contain a 14-carbon ring. These diterpenoids interact with AChRs in complex ways: as irreversible inhibitors at the agonist sites, as noncompetitive inhibitors, or as positive modulators, but no cembranoid was ever shown to have agonistic activity on AChRs. The cembranoid eupalmerin acetate displays positive modulation of agonist-induced currents in the muscle-type AChR and in the related gamma-aminobutyric acid (GABA) type A receptor. Moreover, cembranoids display important biological effects, many of them mediated by nicotinic receptors. Cembranoids from tobacco are neuroprotective through a nicotinic anti-apoptotic mechanism preventing excitotoxic neuronal death which in part could result from anti-inflammatory properties of cembranoids. Moreover, tobacco cembranoids also have anti-inflammatory properties which could enhance their neuroprotective properties. Cembranoids from tobacco affect nicotine-related behavior: they increase the transient initial ataxia caused by first nicotine injection into naive rats and inhibit the expression of locomotor sensitization to repeated injections of nicotine. In addition, cembranoids are known to act as anti-tumor compounds. In conclusion, cembranoids provide a promising source of lead drugs for many clinical areas, including neuroprotection, smoking-cessation, and anti-cancer therapies.

A cembranoid from tobacco prevents the expression of nicotine-induced withdrawal behavior in planarian worms.

Using an adaptation of published behavioral protocols, we determined that acute exposure to the cholinergic compounds nicotine and carbamylcholine decreased planarian motility in a concentration-dependent manner. A tobacco cembranoid (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R-cembranoid), also decreased planarian motility. Experiments in the presence of 1 microM 4R-cembranoid did increase the IC50 for nicotine- but not carbamylcholine-induced decrease in planarian motility. When planarians were exposed for 24 h to either nicotine or carbamylcholine at concentrations near their respective IC50 values and then transferred to plain media, nicotine-exposed, but not carbamylcholine- or cembranoid-exposed worms displayed withdrawal-like distress behaviors. In experiments where planarians were pre-exposed to 100 microM nicotine for 24 h in the presence of 1 microM 4R-cembranoid, the withdrawal-like effects were significantly reduced. These results indicate that the 4R-cembranoid might have valuable applications for tobacco abuse research. This experimental approach using planarians is useful for the initial screening of compounds relevant to drug abuse and dependence.

M2 mutations of the nicotinic acetylcholine receptor increase the potency of the non-competitive inhibitor phencyclidine

Phencyclidine (PCP) is a non‐competitive inhibitor of the nicotinic acetylcholine receptor (nAChR) with biphasic characteristics. At low and high micromolar concentrations, PCP inhibits nAChR from fetal mouse muscle, whereas at intermediate concentrations PCP does not inhibit the receptor. The present study was performed to determine whether the high and low concentration effects of PCP on mouse nAChR were due to interactions of this blocker with channel lining amino acids. In order to test this hypothesis, we examined the ability of PCP to inhibit acetylcholine‐induced currents from wild‐type nAChR and nAChR in which amino acid substitutions were made in the 6′, 8′ and 10′ positions of the M2 transmembrane segments of the receptor. Fetal mouse nAChR from BC3H‐1 cells were expressed in Xenopus laevis oocytes and studied using the two‐electrode voltage clamp technique. The results of this study reveal that in native fetal muscle receptor, PCP potency is not affected by membrane potential between −80 mV and −30 mV. The potency of PCP is increased by mutations in M2 6′, 8′, and 10′ positions. This increase in potency cannot be explained merely by either changes in hydrophobicity/hydrophilicity of amino acids at these positions or by side‐chain size. A model proposing extra‐luminal inhibitory and regulatory sites for PCP explains the lack of voltage‐dependency, the biphasic effect of PCP, and the fact that all M2 mutations increased PCP potency (by disrupting the link with the regulatory sites). J. Neurosci. Res. 61:44–51, 2000.

The ion channel of muscle and electric organ acetylcholine receptors: Differing affinities for noncompetitive inhibitors

Summary1.Muscle and electric organ acetylcholine receptors (AChRs) were expressed inXenopus laevis oocytes and differential effects of noncompetitive blockers on each type of receptor were analyzed using a two-electrode voltage clamp.2.The positively charged channel blockers, phencyclidine (PCP) and tetracaine, displayed a much lower potency on muscle receptor than on the electric organ receptor. The IC50 for both blockers at the elecgrocyte receptor was close to 1µM at -60 mV and even lower at more hyperpolarized voltages. In contrast, with muscle receptor IC50s were 20 to 40µM at -60 or -80 mV.3.Eupalmerin acetate, an uncharged noncompetitive inhibitor that displaces [3H]PCP from its high-affinity binding site, inhibited both receptors with a similar potency: IC50 of 4.9 and 6.4µM for electrocyte and muscle receptors, respectively. However, eupalmerin acetate affected the desensitization process in each receptor type differently and triggered an unusual biphasic response in the muscle receptor.4.These results are discussed with respect to differences in the amino acid sequences of the M2 regions of the two receptors.5.A third type of noncompetitive inhibitor, Mg2+, was also examined and it inhibited both receptors with a similar potency (IC50, 0.5-1.0 mM). However, Mg2+ appeared to act at sites other than the PCP site.

Diterpenoids from caribbean gorgonians act as noncompetitive inhibitors of the nicotinic acetylcholine receptor

Summary1.Three cyclic diterpenoids isolated from gorgonians of theEunicea genus and characterized as eupalmerin acetate (EUAC), 12,13-bisepieupalmerin (BEEP), and eunicin (EUNI) were found to be pharmacologically active on the nicotinic acetylcholine receptor (AChR).2.The receptor from the BC3H-1 muscle cell line was expressed inXenopus laevis oocytes and studied with a two-electrode voltage clamp apparatus.3.All three compounds reversibly inhibited ACh-induced currents, with IC50s from 6 to 35µM. ACh dose-response curves suggested that his inhibition was noncompetitive. The cembranoids also increased the rate of receptor desensitization.4.Radioligand-binding studies using AChR-rich membranes fromTorpedo electric organ indicated that all three cembranoids inhibited high-affinity [3H]phencyclidine binding, with IC50s of 0.8, 11.6, and 63.8µM for EUNI, EUAC, and BEEP, respectively. The cembranoids at a 100µM concentration did not inhibit [α-125I]bungarotoxin binding to either membrane-bound or solubilized AChR.5.It is concluded that these compounds act as noncompetitive inhibitors of peripheral AChR.

Kinin-B2 Receptor Mediated Neuroprotection after NMDA Excitotoxicity Is Reversed in the Presence of Kinin-B1 Receptor Agonists

Background Kinins, with bradykinin and des-Arg9-bradykinin being the most important ones, are pro-inflammatory peptides released after tissue injury including stroke. Although the actions of bradykinin are in general well characterized; it remains controversial whether the effects of bradykinin are beneficial or not. Kinin-B2 receptor activation participates in various physiological processes including hypotension, neurotransmission and neuronal differentiation. The bradykinin metabolite des-Arg9-bradykinin as well as Lys-des-Arg9-bradykinin activates the kinin-B1 receptor known to be expressed under inflammatory conditions. We have investigated the effects of kinin-B1 and B2 receptor activation on N-methyl-D-aspartate (NMDA)-induced excitotoxicity measured as decreased capacity to produce synaptically evoked population spikes in the CA1 area of rat hippocampal slices. Principal Findings Bradykinin at 10 nM and 1 µM concentrations triggered a neuroprotective cascade via kinin-B2 receptor activation which conferred protection against NMDA-induced excitotoxicity. Recovery of population spikes induced by 10 nM bradykinin was completely abolished when the peptide was co-applied with the selective kinin-B2 receptor antagonist HOE-140. Kinin-B2 receptor activation promoted survival of hippocampal neurons via phosphatidylinositol 3-kinase, while MEK/MAPK signaling was not involved in protection against NMDA-evoked excitotoxic effects. However, 100 nM Lys-des-Arg9-bradykinin, a potent kinin-B1 receptor agonist, reversed bradykinin-induced population spike recovery. The inhibition of population spikes recovery was reversed by PD98059, showing that MEK/MAPK was involved in the induction of apoptosis mediated by the B1 receptor. Conclusions Bradykinin exerted protection against NMDA-induced excitotoxicity which is reversed in the presence of a kinin-B1 receptor agonist. As bradykinin is converted to the kinin-B1 receptor metabolite des-Arg9-bradykinin by carboxypeptidases, present in different areas including in brain, our results provide a mechanism for the neuroprotective effect in vitro despite of the deleterious effect observed in vivo.

Tobacco cembranoids block behavioral sensitization to nicotine and inhibit neuronal acetylcholine receptor function.

Cembranoids are cyclic diterpenoids found in tobacco and in marine invertebrates. The present study established that tobacco cembranoids inhibit behavioral sensitization to nicotine in rats and block several types of nicotine acetylcholine receptors (AChRs). 1) At the behavioral level, rat locomotor activity induced by nicotine was significantly increased after seven daily nicotine injections. This sensitization to nicotine was blocked by mecamylamine (1 mg/kg) and by the cembranoids eunicin, eupalmerin acetate (EUAC), and (4R)‐2,7,11‐cembratriene‐4‐6‐diol (4R), each at 6 mg/kg. None of these compounds modified locomotor activity of nonsensitized rats. 2) In cells expressing human AChRs, cembranoids blocked carbamoylcholine‐induced 86Rb+ flux with IC50 in the low micromolar range. The cell lines used were the SH‐EP1‐hα4β2 cell line heterologously expressing human α4β2‐AChR, the SH‐SY5Y neuroblastoma line naturally expressing human ganglionic α3β4‐AChR, and the TE671/RD cell line naturally expressing embryonic muscle α1β1γδ‐AChR. The tobacco cembranoids tested were 4R and its diastereoisomer 4S, and marine cembranoids tested were EUAC and 12,13‐bisepieupalmerin. 3) At the molecular level, tobacco (4R and 4S) and marine (EUAC) cembranoids blocked binding of the noncompetitive inhibitor [3H]tenocyclidine to AChR from Torpedo californica electric organ. IC50 values were in the submicromolar to low‐micromolar range, with 4R displaying an order of magnitude higher potency than its diastereoisomer, 4S. J. Neurosci. Res. 64:18–25, 2001.

Spermine increases paired-pulse facilitation in area CA1 of hippocampus in a calcium-dependent manner

The effect of spermine on neurotransmission was studied in area CA1 of the hippocampal slice preparation. Paired-pulse stimulation (20 ms interpulse interval) was delivered to stratum radiatum; the evoked field potential responses were recorded simultaneously from stratum radiatum and from stratum pyramidale. At mM and sub-mM concentrations, spermine decreased the slope of pEPSP in stratum radiatum and the area of the conditioning population spike in stratum pyramidale. Short-latency paired-pulse inhibition of the population spike was converted to facilitation by spermine. These effects of spermine resembled those observed at low calcium concentration. In addition, dose-response and input-output curves determined at various Ca2+ concentrations demonstrated that the depressant effects of spermine were larger at low Ca2+ levels. The results support the notion that spermine competitively blocks presynaptic voltage-sensitive Ca2+ channels, thus causing a decreased release of neurotransmitter. Since spermine is present in brain, it is likely that it is a natural modulator of Ca2+ channels.

4R-cembranoid protects against diisopropylfluorophosphate-mediated neurodegeneration.

Many organophosphorous esters synthesized for applications in industry, agriculture, or warfare irreversibly inhibit acetylcholinesterase, and acute poisoning with these compounds causes life-threatening cholinergic overstimulation. Following classical emergency treatment with atropine, an oxime, and a benzodiazepine, surviving victims often suffer brain neurodegeneration. Currently, there is no pharmacological treatment to prevent this brain injury. Here we show that a cyclic diterpenoid, (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R) ameliorates the damage caused by diisopropylfluorophosphate (DFP) in the hippocampal area CA1. DFP has been frequently used as a surrogate for the warfare nerve agent sarin. In rats, DFP is lethal at the dose used to cause brain damage. Therefore, to observe brain damage in survivors, the death rate was reduced by pre-administration of the peripherally acting antidotes pyridostigmine and methyl atropine or its analog ipratropium. Pyridostigmine bromide, methyl atropine nitrate, and ipratropium bromide were dissolved in saline and injected intramuscularly at 0.1mg/kg, 20mg/kg, and 23mg/kg, respectively. DFP (9mg/kg) dissolved in cold water was injected intraperitoneally. 4R (6mg/kg) dissolved in DMSO was injected subcutaneously, either 1h before or 5 or 24h after DFP. Neurodegeneration was assessed with Fluoro-Jade B and amino cupric silver staining; neuroinflammation was measured by the expression of nestin, a marker of activated astrocytes. Forty-eight hours after DFP administration, 4R decreased the number of dead neurons by half when injected before or after DFP. 4R also significantly decreased the number of activated astrocytes. These data suggest that 4R is a promising new drug that could change the therapeutic paradigm for acute poisoning with organophosphorous compounds by the implementation of a second-stage intervention after the classical countermeasure treatment.

Spermine depresses NMDA, K/AMPA and GABAA-mediated synaptic transmission in the rat hippocampal slice preparation

The effects of spermine, an endogenous polyamine, were examined in area CA1 of the rat hippocampal slice preparation. Spermine, at low millimolar concentrations, rapidly and potently depressed NMDA and K/AMPA-mediated population EPSPs, and GABA-mediated monosynaptic population IPSPs. These effects contrast with its well-known potentiation of NMDA currents at lower concentrations. Our results raise the possibility that the large intracellular stores of spermine that are released after various neural insults could act as an endogenous neuroprotective mechanism by limiting excessive calcium entry.