Structure–activity studies with ring E analogues of methyllycaconitine on bovine adrenal α3β4* nicotinic receptors
Introduction
Nicotinic acetylcholine receptors (nAChRs) containing α3 subunits are found on postsynaptic neurons in autonomic ganglia and chromaffin cells of the adrenal medulla. The nAChR subtypes believed to be expressed in these tissues are α3β4*, α3α5β4 and α3α5β2β4 (Lukas et al., 1999). These α3-containing nAChRs play a prominent role in autonomic neurotransmission and adaptive responses to stress. α3-Containing nAChRs have also been found in several brain regions (e.g. substantia nigra, ventral tegmental area, hippocampus, the medial habenula and interpeduncular nucleus) (for reviews, see Jones et al., 1999, Picciotto et al., 2000, Cordero-Erausquin et al., 2000). The precise subunit composition and the physiological role of these nAChRs in the CNS remain to be elucidated, but have been reported to be involved with the control of norepinephrine (Sershen et al., 1997) and dopamine release (Kulak et al., 1997). A diminution of α3 mRNA in regions of the aged brain of humans (Terzano et al., 1998) and rats (Charpantier et al., 1999) has also been reported, suggesting a contribution of neurons expressing α3-containing nAChRs to age associated memory impairment.
Subtypes of neuronal nAChRs can be pharmacologically differentiated by a variety of agents found in nature, including snake venom neurotoxins (Luetje et al., 1990), marine snail conotoxins (McIntosh et al., 1994, Johnson et al., 1995, Cartier et al., 1996), and the plant alkaloid, methyllycaconitine (MLA) (Macallan et al., 1988, Ward et al., 1990). MLA is a potent, nonpeptide, nAChR antagonist showing selectivity for α7 nAChRs. MLA also interacts with other nAChRs subtypes, albeit at lower affinity (Yum et al., 1996, Davies et al., 1999). Recently, our laboratory reported that MLA inhibits nAChR-stimulated adrenal catecholamine secretion (Bergmeier et al., 1999) demonstrating activity of MLA on α3β4* nAChRs. These effects, though, occur at MLA concentrations that are approximately 1000-fold higher than concentrations that affect α7 nAChRs.
Although, the nonpeptide nature of MLA lends itself to structure–activity relationship (SAR) studies, few SAR studies have been reported and are focused on α7 nAChR activity (Davies et al., 1997, Hardick et al., 1996). Our laboratory has recently described simple ring E analogues of MLA containing the succinimidoylanthranilate side chain (Fig. 1). The availability of these analogues allows for the study of structural determinants of MLA which may be important for its activity on bovine α3β4* nAChRs. The demonstration that the ring E analogues inhibit nAChR-stimulated adrenal catecholamine secretion (Bergmeier et al., 1999) suggests that the ring E moiety of MLA containing the succinimidoylanthranilate side chain may be important for the activity of MLA on adrenal nAChRs. The following studies were designed to further characterize the SAR of ring E analogues of MLA to assess structural determinants affecting activity on adrenal nAChRs.
Section snippets
Materials
Ring E analogues (LB-1 to LB-8, IB-1, AB-2) were synthesized as described by Bergmeier et al. (1999). Limited availability of certain analogues due to difficulties in synthesis prevented their use in some studies. (−)Nicotine hydrogen tartrate, α-bungarotoxin (αBGT), polyethyleneimine (PEI) and components of N2+ media were obtained from the Sigma Chemical Company (St. Louis, MO). Dulbecco's Modified Eagle Medium (DMEM) and DMEM/F12 (used in N2+ medium) were obtained from Life Technologies
Results
MLA has been reported to inhibit bovine adrenal catecholamine secretion mediated by both α7 nAChRs (IC50 value, 0.1 μM) (Lopez et al., 1998) and α3β4* nAChRs (low micromolar) (Bergmeier et al., 1999). Our laboratory has previously identified ring E analogues of MLA that also inhibit nicotine–stimulated adrenal catecholamine secretion (Bergmeier et al., 1999), implicating activity of these analogues on α3β4* nAChRs. In the following studies the concentration–response effects of several ring E
Discussion
The principal receptors that mediate adrenal catecholamine secretion are believed to be α3β4* nAChRs (Wenger et al., 1997). Our laboratory has previously identified ring E analogues of MLA that inhibit nicotine–stimulated adrenal catecholamine secretion (Bergmeier et al., 1999), implicating activity of these analogues on α3β4* nAChRs. The N-phenpropyl analogue (LB-8) was the most potent analogue with an IC50 value (11 μM), similar in potency to other inhibitors of adrenal catecholamine release
Acknowledgements
This project was supported by NIH Grants DA12707 (SCB, DBM) and DA10569 (DBM). DLB was supported as a NIDA Underrepresented Minority Supplement Awardee (DA10569). KIA was supported by a fellowship from the Egyptian Cultural and Educational Bureau. RBF was supported by an NIH Training grant (MH19936).
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