Effects of piperine, the pungent component of black pepper, at the human vanilloid receptor (TRPV1).

1. We have characterised the effects of piperine, a pungent alkaloid found in black pepper, on the human vanilloid receptor TRPV1 using whole-cell patch-clamp electrophysiology. 2. Piperine produced a clear agonist activity at the human TRPV1 receptor yielding rapidly activating whole-cell currents that were antagonised by the competitive TRPV1 antagonist capsazepine and the non-competitive TRPV1 blocker ruthenium red. 3. The current-voltage relationship of piperine-activated currents showed pronounced outward rectification (25+/-4-fold between -70 and +70 mV) and a reversal potential of 0.0+/-0.4 mV, which was indistinguishable from that of the prototypical TRPV1 agonist capsaicin. 4. Although piperine was a less potent agonist (EC50=37.9+/-1.9 microM) than capsaicin (EC50=0.29+/-0.05 microM), it demonstrated a much greater efficacy (approximately two-fold) at TRPV1. 5. This difference in efficacy did not appear to be related to the proton-mediated regulation of the receptor since a similar degree of potentiation was observed for responses evoked by piperine (230+/-20%, n=11) or capsaicin (284+/-32%, n=8) upon acidification to pH 6.5. 6. The effects of piperine upon receptor desensitisation were also unable to explain this effect since piperine resulted in more pronounced macroscopic desensitisation (t(1/2)=9.9+/-0.7 s) than capsaicin (t(1/2)>20 s) and also caused greater tachyphylaxis in response to repetitive agonist applications. 7. Overall, our data suggest that the effects of piperine at human TRPV1 are similar to those of capsaicin except for its propensity to induce greater receptor desensitisation and, rather remarkably, exhibit a greater efficacy than capsaicin itself. These results may provide insight into the TRPV1-mediated effects of piperine on gastrointestinal function.

Comparative phenotypic resolution of spontaneous, D2-like and D1-like agonist-induced orofacial movement topographies in congenic mutants with dopamine D2 vs. D3 receptor "knockout".

Using a novel system, the role of D2-like dopamine receptors in distinct topographies of orofacial movement was assessed in mutant mice with congenic D2 vs. D3 receptor knockout, and compared with findings in D1A mutants. Under spontaneous conditions, D2 mutants evidenced increased vertical jaw movements and unaltered horizontal jaw movements, with reductions in tongue protrusions and incisor chattering; in D3 mutants, only incisor chattering was reduced. Given previous evidence that D1A mutants show reduced horizontal but not vertical jaw movements, this indicates that apparent oppositional D1-like:D2-like interactions in the regulation of composited jaw movements may in fact reflect the independent actions of D2 receptors to inhibit vertical jaw movements and of D1A receptors to facilitate horizontal jaw movements. Effects of the D2-like agonist RU 24213 to exert greater reduction in horizontal than in vertical jaw movements were not altered prominently in either D2 or D3 mutants. The D1-like agonists A 68930 and SK&F 83959 induced vertical jaw movements, tongue protrusions, and incisor chattering; induction of tongue protrusions by A 68930 was reduced in D2 mutants. D2 receptors exert topographically specific regulation of orofacial movements in a manner distinct from their D1A counterparts, while D3 receptors exert only minor regulation of such movements.

Topographical Assessment of Ethological and Dopamine Receptor Agonist-Induced Behavioral Phenotype in Mutants with Congenic DARPP-32 'Knockout'.

Congenic (10 backcrosses into C57BL/6J) mutants with targeted gene deletion of DARPP-32, a neuronal phosphoprotein regarded as an essential mediator of the biological effects of dopamine (DA), were assessed phenotypically using an ethologically based approach that resolves all topographies of behavior in the mouse repertoire. Over initial exploration, female, but not male, DARPP-32 mutants evidenced increased locomotion and decreased grooming, while a decrease in rearing seated was evident in mutants of both genders; continuing assessment over several hours did not reveal additional phenotypic effects. Following challenge with the nonselective DA receptor agonist apomorphine, low doses were associated with reduced levels of sniffing, grooming, total rearing, and rearing seated in DARPP-32 mutants relative to wildtypes; this would suggest some role for DARPP-32 in mediating the biological effects of presynaptic D(2)-like autoreceptor or inhibitory postsynaptic D(2)-like receptor activation. Following challenge with higher doses, while stereotyped sniffing and locomotion with chewing was largely unaltered, the additional murine response of Straub tail was essentially abolished in DARPP-32 mutants, indicating some specific involvement of DARPP-32 in mediating this topography of behavior; additionally, there were overall reductions in levels of sniffing, total rearing, rearing seated, and grooming in DARPP-32 mutants that were unrelated to the dose of apomorphine administered, indicating broader topographical effects following the stress of the injection procedure relative to more naturalistic conditions. The developmental absence of DARPP-32 following targeted gene deletion appears to be associated with compensatory processes that maintain certain topographies of spontaneous and agonist-induced DAergic function, while other topographies remain impaired.

Congenic D1A dopamine receptor mutants: ethologically based resolution of behavioural topography indicates genetic background as a determinant of knockout phenotype.

D(1A)-null mice were backcrossed over 14 generations into a C57BL/6 background to result in essential elimination (to <0.005%) of any contribution from the 129/Sv component of their initially mixed (129/SvxC57BL/6) background. Their phenotype was assessed using an ethologically based approach that resolves each individual topography of behaviour in the natural repertoire. Habituation of sniffing, locomotion, rearing seated, and rearing to wall in wild types over several hours was profoundly retarded in congenic D(1A) mutants; conversely, rearing free and sifting were essentially abolished. Resultant increases in individual topographies of behaviour were substantially greater in congenic D(1A) mutants than in those on a mixed background. This phenotype was little altered by the selective D(1)-like antagonist SCH 23390 and could not be blocked by the selective D(2)-like antagonist YM 09151-2. The selective D(1)-like agonist SK&F 83959 could not further elevate those behaviours already heightened in congenic D(1A) mutants, while the induction of stereotyped sniffing and plodding locomotion by the selective D(2)-like agonist RU 24213 was disrupted. Genetic background appears to modulate critically the magnitude but not the general nature of the D(1A)-null phenotype, which may involve compensatory processes independent of other D(1)-like or D(2)-like receptors.

Phenotypic, ethologically based resolution of spontaneous and D(2)-like vs D(1)-like agonist-induced behavioural topography in mice with congenic D(3) dopamine receptor "knockout".

Uncertainty as to the functional role of the D(3) dopamine receptor, due primarily to a paucity of selective agonists or antagonists, is being addressed in mice with targeted gene deletion ("knockout") thereof. This study describes, for the first time, the phenotype of congenic D(3)-null mice. Initially, 129/Sv x C57BL/6 D(3)-null mice were backcrossed 14 times onto C57BL/6; they were then assessed using an ethologically based approach which resolves all topographies of behaviour within the mouse repertoire. The ethogram of D(3)-null mice, on comparison with wildtypes, was characterised by no alteration in any topography of behaviour over an initial period of exploration; subsequent assessment over several hours revealed only increased rearing among females due to delayed habituation. Low doses of the selective D(2)-like agonist RU 24213 (0.016-0.25 mg/kg) inhibited topographies of exploratory behaviour; this effect was diminished in D(3)-null mice only when investigated following prolonged habituation, and then only for certain topographies of behaviour, primarily sniffing and rearing. High doses of RU 24213 (0.1-12.5 mg/kg) induced stereotyped sniffing and "ponderous" locomotion, while the selective D(1)-like agonist SK&F 83959 (0.016-2.0 mg/kg) promoted characteristic grooming syntax; these effects did not differ materially between the genotypes. When examined topographically on an essentially congenic C57BL/6 background (<0.005% 129/Sv), the resultant phenotype indicated essential conservation of the mouse ethogram, high-dose D(2)-like stimulatory effects, and D(1)-like stimulatory effects in the absence of D(3) receptors. A role for D(3) receptors in inhibitory processes appeared topographically circumscribed and only when baseline levels of behaviour were low.