PPAHV

Identification of species-specific determinants of the action of the antagonist capsazepine and the agonist PPAHV on TRPV1.[Pubmed:14960593]

J Biol Chem. 2004 Apr 23;279(17):17165-72.

The vanilloid receptor 1 (VR1 or TRPV1) ion channel is activated by noxious heat, low pH and by a variety of vanilloid-related compounds. The antagonist, capsazepine is more effective at inhibiting the human TRPV1 response to pH 5.5 than the rat TRPV1 response to this stimulus. Mutation of rat TRPV1 at three positions in the S3 to S4 region, to the corresponding human amino acid residues I514M, V518L, and M547L decreased the IC(50) values for capsazepine inhibition of the pH 5.5 response from >10,000 nm to 924 +/- 241 nm in [Ca(2+)](i) assays and increased capsazepine inhibition of the capsaicin response to levels seen for human TRPV1. We have previously noted that phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) is a strong agonist of rat TRPV1 but not human TRPV1 in [Ca(2+)](i) assays (1). Mutation of methionine 547 in S4 of rat TRPV1 to leucine, found in human TRPV1 (M547L), reduced the ability of PPAHV to activate TRPV1 by approximately 20-fold. The reciprocal mutation of human TRPV1 (L547M) enabled the human receptor to respond to PPAHV. These mutations did not significantly affect the agonist activity of capsaicin, resiniferatoxin (RTX) or olvanil in [Ca(2+)](i) assays. Introducing the equivalent mutation into guinea pig TRPV1 (L549M) increased the agonist potency of PPAHV by > 10-fold in the [Ca(2+)](i) assay and increased the amplitude of the evoked current. The rat M547L mutation reduced the affinity of RTX binding. Thus, amino acids within the S2-S4 region are important sites of agonist and antagonist interaction with TRPV1.

Functional and desensitizing effects of the novel synthetic vanilloid-like agent 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) in the perfused rat hindlimb.[Pubmed:11090114]

Br J Pharmacol. 2000 Dec;131(7):1408-12.

1. In the present study, the effects of the novel vanilloid agonist, 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), on oxygen consumption (VO(2)) and vascular resistance (perfusion pressure, PP) were investigated in the constant flow, perfused rat hindlimb. The acute desensitizing properties of this novel synthetic agent were also examined. 2. Maximum stimulation of VO(2) was produced by 0.2 microM PPAHV (delta VO(2), 0.83+/-0.06 micromol g(-1) h(-1)) and was accompanied by mild vasoconstriction (increase in PP; 8.0+/-1.1 mmHg). The highest concentration of PPAHV tested (2 microM) caused inhibition of VO(2) (delta VO(2), -2.73+/-0.51 micromol g(-1) h(-1)) and strong vasoconstriction (delta PP, 42.0+/-1.2 mmHg). 3. Capsazepine (10 microM) caused a parallel shift to the right of both VO(2) and PP concentration-response curves for PPAHV (pK(b)=5.00), indicative of competitive binding to vanilloid receptors. 4. The stimulation of VO(2) produced by 0.2 microM PPAHV decreased, but was not completely abolished, after repeated infusion of PPAHV (change in VO(2), first infusion, 0.66+/-0.18 micromol g(-1) h(-1); sixth infusion, 0.29+/-0. 08 micromol g(-1) h(-1), P<0.05), an acute tachyphylactic response not previously seen with the repeated infusion of other vanilloid analogues. Conversely, the PP response to repeated PPAHV infusion increased (delta PP, first infusion, 5.8+/-0.7 mmHg; sixth infusion, 9.0+/-0.6 mmHg, P<0.05). 5. In conclusion, PPAHV produces vasoconstriction and a biphasic effect on VO(2) in the perfused rat hindlimb very similar to that induced by naturally occurring vanilloids. Both effects are blocked by the competitive antagonist capsazepine. Since, the metabolic response to low concentrations of PPAHV (stimulation of VO(2)) undergoes tachyphylaxis, the present data suggest that PPAHV desensitizes putative vanilloid receptors in the hindlimb.

Phorboid 20-homovanillates induce apoptosis through a VR1-independent mechanism.[Pubmed:10903936]

Chem Biol. 2000 Jul;7(7):483-92.

BACKGROUND: Vanilloids, such as capsaicin and resiniferatoxin (RTX), are recognized at the cell surface by vanilloid receptor type 1 (VR1), which has recently been cloned. VR1 mediates the effects of capsaicin and RTX in VR1-expressing cells, but vanilloids can induce apoptosis through a pathway not mediated by VR1. Phorboid 20-homovanillates can be used to investigate cell death induced by vanilloids. RESULTS: 12,13-Diacylphorbol-20 homovanillates were prepared by the sequential esterification of the natural polyol. Phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) induced apoptosis in Jurkat cells to the same extent as RTX. Apoptosis was preceded by an increase in intracellular reactive oxygen species and by the loss of mitochondrial transmembrane potential. PPAHV-induced apoptosis was mediated by a pathway involving caspase-3 activation and was initiated at the S phase of the cell cycle. The cell-death pathway triggered by VR1 activation was studied in 293T cells transfected with the cloned rat vanilloid receptor. In this system, capsaicin and PPAHV induced cell death by an apparent necrotic mechanism, which was selectively inhibited by the competitive vanilloid receptor antagonist capsazepine. Interestingly, phorbol-12, 13-bisnonanoate-20-homovanillate, an analogue of PPAHV, induced cell death in VR1-transfected cells but could not trigger apoptosis in the Jurkat cell line. CONCLUSIONS: Vanilloids can induce cell death through different signalling pathways. The cell death induced in a VR1-independent manner has the hallmark of apoptosis, whereas the cell death mediated by vanilloids binding to VR1 is seemingly necrotic. Phorboid homovanillates that have antitumour and anti-inflammatory activities but lack the undesirable side effects of the natural vanilloids could be developed as potential drugs.

A novel agonist, phorbol 12-phenylacetate 13-acetate 20-homovanillate, abolishes positive cooperativity of binding by the vanilloid receptor.[Pubmed:8901026]

Eur J Pharmacol. 1996 Mar 28;299(1-3):221-8.

Capsaicin binds to a specific recognition site, referred to as the vanilloid receptor, which it shares with the natural, ultrapotent agonist resiniferatoxin and with the competitive antagonist capsazepine. Upon binding to its receptor, capsaicin opens a cation channel leading to Ca2+ influx. The binding of capsaicin or resiniferatoxin by the vanilloid receptor follows a sigmoidal saturation curve, indicative of positive cooperativity. The biological significance of this positive cooperative behaviour is unknown, as is the mechanism responsible for it. We have developed a novel ligand, phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), which binds to cultured rat sensory neurons (with a Ki of 3.1 +/- 0.4 microM), and induces Ca2+ uptake by them (with an ED50 of 1.8 +/- 0.3 microM) with similar affinities and in a non-cooperative manner (Hill coefficients are 0.99 and 1.06 for binding and Ca2+ uptake, respectively). The behaviour of PPAHV thus contrasts with resiniferatoxin or capsaicin not only in the lack of cooperativity but also in the relative potencies for resiniferatoxin binding versus Ca2+ uptake (resiniferatoxin is less potent and capsaicin is more potent for induction of Ca2+ uptake than for binding). In further experiments in which the concentration of [3H]resiniferatoxin was varied, 1 microM PPAHV likewise reduced the cooperativity index that characterizes resiniferatoxin binding to rat spinal cord membranes from 2.3 +/- 0.1 to 1.1 +/- 0.2; in parallel experiments, neither capsaicin nor capsazepine (both at a concentration of 2 microM) affected binding cooperativity. Moreover, PPAHV (1 microM) turned the bi-phasic dissociation curve of resiniferatoxin into a monophasic curve, eliminating the second, slow-dissociation phase. The present results suggest that positive cooperativity is a ligand-induced feature rather than an inherent property of vanilloid receptors. A comparison of the spectrum of biological activity of ligands which bind to vanilloid receptors with different degrees of cooperativity may provide an approach to explore the functional significance of this binding behaviour.