Modulatory Effects of Eschscholzia californica Alkaloids on Recombinant GABAA Receptors

Abstract

The California poppy (Eschscholzia californica Cham.) contains a variety of natural compounds including several alkaloids found exclusively in this plant. Because of the sedative, anxiolytic, and analgesic effects, this herb is currently sold in pharmacies in many countries. However, our understanding of these biological effects at the molecular level is still lacking. Alkaloids detected in E. californica could be hypothesized to act at GABAA receptors, which are widely expressed in the brain mainly at the inhibitory interneurons. Electrophysiological studies on a recombinant α 1 β 2 γ 2 GABAA receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 μM. However, (S)-reticuline behaved as positive allosteric modulator at the α 3, α 5, and α 6 isoforms of GABAA receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by (S)-reticuline at the α 3 β 2 γ 2 and α 5 β 2 γ 2 GABAA receptors. Interestingly, α 1, α 3, and α 5 were not significantly affected by (R)-reticuline, 1,2-tetrahydroreticuline, codeine, and morphine-suspected (S)-reticuline metabolites in the rodent brain.

Figures

Figure 1

Alkaloids identified in the aerial parts of Eschscholzia californica.

Figure 2

(a) LC UV chromatogram of Eschscholzia californica extract before fractionation. Peak identification: (1) reticuline; (2) caryachine; (3) N-methyllaurotetanine; (4) protopine; (5) californidine; (6) allocryptopine; (7) escholtzine; (8) sanguinarine; and (9) chelerythrine. (b) LC-MS trace of “NMT sample” containing 82% N-methyllaurotetanine, 10% reticuline, and 8% caryachine with traces of other alkaloids.

Figure 3

(a) (A) Electrospray ESI trace and (B) tandem MS/MS fragmentation pattern for selected ion m/z 354 corresponding to alkaloid protopine detected in a pharmacy capsule (300 mg) of E. californica (AMM 57426001) following alkaloid extraction and separation (Method A). (b) Electrospray ESI trace (A) and tandem MS/MS profile (B) for 6S,12S-neocaryachine-7-O-methyl ether N-metho salt detected in the same AMM 57426001 sample. Note that m/z 354 for this pavine alkaloid is practically identical to that of protopine; however, it gives quite different fragmentation pattern.

Figure 4

Modulation of recombinant GABAA receptors of different subunit composition by (S)-reticuline. Receptors were expressed in Xenopus oocytes and concentration dependent modulation was determined using electrophysiological techniques. Each curve is the average of three to four determinations (data points ± standard deviation).

Figure 5

Effect of a benzodiazepine antagonist on the potentiation of α5β2γ2 GABAA receptors by (S)-reticuline. Application of 0.5 μM GABA was followed by application of 0.5 μM GABA/10 μM (S)-reticuline and subsequently by 0.5 μM GABA/10 μM (S)-reticuline/1 μM Ro15-1788. Providing (S)-reticuline would act at the benzodiazepine site elicited currents would be expected to go back to the size elicited by GABA alone. Instead, a potentiation was observed. Similar observations were made in two additional experiments.