Kinetics of Penetration Influence the Apparent Potency of Vanilloids on TRPV1

Abstract

Evidence that the ligand binding site of TRPV1 lies on the inner face of the plasma membrane and that much of the TRPV1 itself is localized to internal membranes suggests that the rate of ligand entry into the cell may be an important determinant of the kinetics of ligand action. In this study, we synthesized a BODIPY TR-labeled fluorescent capsaicin analog (CHK-884) so that we could directly measure ligand entry. We report that CHK-884 penetrated only slowly into Chinese hamster ovary (CHO) cells expressing rat TRPV1, with a t 1/2 of 30 Ϯ 4 min, and localized in the endoplasmic reticulum and Golgi. Although CHK-884 was only weakly potent for TRPV1 binding (K i ϭ 6400 Ϯ 230 nM), it was appreciably more potent when assayed by intracellular calcium imaging and was 3.2-fold more potent with a 1-h incubation time (37 nM) than with a 5-min incubation time. Olvanil, a highly lipophilic vanilloid, yielded an EC 50 of 4.3 nM upon intracellular calcium imaging with an incubation time of 1 h, compared with an EC 50 value of 29.5 nM for calcium imaging assayed at 5 min. Likewise, the antagonist 5-iodoresiniferatoxin (5-iodo-RTX) displayed a K i of 4.2 pM if incubated with CHO-TRPV1 cells for 2 h before addition of capsaicin compared with 1.5 nM if added simultaneously. We conclude that some vanilloids may have slow kinetics of uptake; this slow uptake may affect assessment of structure activity relations and may represent a significant factor for vanilloid drug design.TRPV1 is a central nociceptor mediating response to vanilloids (such as capsaicin and RTX), heat, low pH, and endogenous ligands (Szallasi and Blumberg, 1989;Caterina et al., 1997;Zygmunt et al., 1999;Hwang et al., 2000;Gavva et al., 2004). In addition, it has a prominent role in the functioning of C-fiber sensory neurons, thus becoming a promising therapeutic target for chronic pain, bladder hyperreflexia, pruritus, diabetic neuropathy, postherpetic neuropathy, or cough (Robbins, 2000;Morice and Geppetti, 2004).In this study, we were concerned with the influence of the rate of vanilloid penetration into the cell on apparent vanilloid activity. It is clear that TRPV1 shows complicated cellular localization. Contrary to the expectation that TRPV1 should be localized at the plasma membrane, most seems to be located at internal membranes (Olah et al., 2001). Consistent with this pattern of localization, multiple research groups have shown that TRPV1 can function to release calcium from endoplasmic stores as well as permit calcium entry from outside the cell (Eun et al., 2001;Marshall et al., 2003). Vanilloids obviously need to penetrate into the cell to gain access to the endoplasmic reticulum localized TRPV1.For the TRPV1 located at the plasma membrane, the original view was, likewise, that the vanilloid binding site of -2004-000-10132-0

Kunitz-Type Peptide HCRG21 from the Sea Anemone Heteractis crispa Is a Full Antagonist of the TRPV1 Receptor

“…Furthermore, a model of slow interaction also agrees with the data regarding kinetics of vanilloids penetration into the cell (equilibrium is reached near to 2 h) and herewith a dramatic decreasing of effective ligand concentration (by two orders of magnitude at 2 h of pre-incubation conditions) when compared to simultaneous application [62]. …”

Endocannabinoids Modulate Human Epidermal Keratinocyte Proliferation and Survival via the Sequential Engagement of Cannabinoid Receptor-1 and Transient Receptor Potential Vanilloid-1

“…(iii) The effect of AEA to increase [Ca 2 þ ] i was realized only after a long-term incubation of the cells, in contrast to the immediate action of the ''direct'' TRPV1 agonist CAPS. Although we cannot exclude the possibility that anandamide exhibited a slower onset of action due to its higher lipophilicity, as compared with that of CAPS (as nicely shown by Lazar et al, 2006 andUrsu et al, 2010), these results suggest that AEA may not directly activate TRPV1, but rather multiple (and yet to be determined) AEA-evoked signaling pathways are involved in the opening of the ion channel. (iv) That these ''upstream'' mechanisms involve the preceding action of AEA on CB 1 is supported by the fact that, whereas both CB1 and TRPV1 antagonists were able to equally suppress the amplitude of the AEA-induced [Ca 2 þ ] i elevations, the CB 1 antagonist AM-251 (unlike the ''channel antagonists'' of TRPV1 that do not affect the activity of CB 1 ) markedly increased the already very long TTP value of the [Ca 2 þ ] i transients.…”

Differential modulation of agonist and antagonist structure activity relations for rat TRPV1 by cyclosporin A and other protein phosphatase inhibitors

“…Toth et al (2005), for example, highlighted differences in the rate of initial response to different ligands, probably reflecting the potential slow penetration of some compounds into cells (Lazar et al, 2006), differences in the kinetics of desensitization, and differences in the pattern of response of individual cells. Mohapatra and Nau (2005) have suggested an effect of cyclosporin A treatment on reduced desensitization in response to capsaicin.…”