The first finding of this study is that the blockade of TRPV1 activation in the heat mode does not contribute to the development of TRPV1 antagonist-induced hyperthermia. Indeed, the graphical analysis has found no correspondence between the potency of an antagonist to cause hyperthermia and its potency to block the heat mode (), whereas the mathematical model has shown that the contribution of the blockade of the heat mode to the hyperthermic effect is ~0 (). That the heat mode is uninvolved agrees with our study with AMG0347, in which no positive correlation was found between the body temperature at the time of drug administration and the magnitude of the hyperthermic response, thus suggesting that TRPV1 channels that mediate AMG0347-induced hyperthermia are activated by nonthermal signals (Steiner et al., 2007
Our second finding is that the potency of TRPV1 antagonists to cause hyperthermia relates most closely to their potency to block TRPV1 activation by protons. The importance of the proton mode was proposed earlier, based on an observation that AMG8562, an antagonist that potentiates TRPV1 activation by protons, did not cause hyperthermia in rats (Lehto et al., 2008
). The present work determines the quantitative contribution of the proton mode. The sensitivity of the hyperthermic response to the blockade in this mode is the highest (0.43-0.65), regardless of which set of data is fit into the model, and it accounts for 45-81% of the variance of the thermoregulatory effect of TRPV1 antagonists (). Both the graphical () and comparative () analyses confirm the principal role of the proton mode. Guinea pigs, the species in which CPZ blocks TRPV1 activation by protons, are more sensitive to the hyperthermic effect of this antagonist than rats, the species in which CPZ does not block the proton mode.
Our third finding is that the blockade of the CAP mode either does not contribute to TRPV1 antagonist-induced hyperthermia or makes only a limited contribution. Depending on which data set is fit into the model, the sensitivity of the hyperthermic response to the blockade of this mode is either negligible or moderate ().
Based on these three findings, the hyperthermic response to TRPV1 antagonists is triggered by the blockade of the proton mode of TRPV1 activation, either alone or together with the CAP mode. Hence, it is likely that the body temperature is tonically suppressed by a low tissue pH, either alone or together with endogenous ligands. Because TRPV1 antagonists cause hyperthermia by acting within the abdomen (Steiner et al., 2007
; McGaraughty et al., 2009
), it is of interest that two major intra-abdominal organs, the stomach and colon, have an acidic environment (Holzer, 2007
). TRPV1 channels are abundant on dorsal-root and nodose afferents innervating these organs and serve there as acidity sensors (Holzer, 2007
; Sugiura et al., 2007
). By acting on two different extracellular residues, the pH has a dual effect on the TRPV1 channel: at a low pH (< 6), protons act as agonists and open the channel; at a higher pH (6-7), protons lower the threshold for TRPV1 activation (Jordt et al., 2000
). One of these ligands, oleoylethanolamide (OEA), is present in the intestinal wall at concentrations comparable with its EC50
against TRPV1 (Fu et al., 2003
). At least some responses to OEA are ablated in CAP-desensitized animals, thus suggesting an action on TRPV1-expressing afferent fibers (Rodriguez de Fonseca et al., 2001
). OEA also causes hypothermia in mice (Watanabe et al., 1999
). Although TRPV1 activation by protons alone or in conjunction with lipid mediators is a plausible scenario, other scenarios are also possible. For example, multiple cationic stimuli (e.g.
, and polycations such as spermine) can directly gate or sensitize the TRPV1 channel by interacting with the same sites in the pore loop domain that are the targets for protons (Ahern et al., 2005
; Ahern et al., 2006
Potential TRPV1 antagonists are routinely screened by pharmaceutical companies in three main modes (heat, proton, and CAP), and the corresponding IC50
data are available for a large number of compounds (see, e.g.
, ). Hence, the goal of the present study was to find a general rule for identifying hyperthermia-free TRPV1 antagonists based on standard in vitro
tests in the three modes. The TRPV1 channel can also be activated independently of the three main modes, e.g.
, by ammonia or intracellular alkalization (Dhaka et al., 2009
). The potentiation effect of modest acidity on vanilloid-evoked TRPV1 activation also has a different substrate than the activation effect exerted by high concentrations of protons (Jordt et al., 2000
). How the potency of TRPV1 antagonists to block these, less studied, modes correlates with the potency to cause hyperthermia remains unknown.
As for the main modes of TRPV1 activation, our study shows that hyperthermia-free TRPV1 antagonists do not block the proton mode, even if they potently block the heat mode, and that decreasing the potency to block the CAP mode of TRPV1 activation may further decrease the potency to cause hyperthermia. This profile identified is similar to the profile of CPZ, the first relatively selective TRPV1 antagonist synthesized, against the rat or murine TRPV1 channel (). Indeed, although CPZ has been used extensively in thermoregulation studies in rats and mice (Dogan et al., 2004
; Shimizu et al., 2005
), it has not been reported to cause hyperthermia. On the other hand, analgesic efficacy for CPZ has been demonstrated in rat and murine models of chemically induced hyperalgesia, inflammatory pain, and neurogenic inflammation (Walker et al., 2003
; Dinis et al., 2004
; Hutter et al., 2005
), even though CPZ did not cause analgesia in some rat models in the study by Walker et al. (2003)
Because eliminating the hyperthermic side effect requires a drastic decrease in the potency to block TRPV1 activation by protons and possibly by CAP, it is important to determine under which conditions the blockade of thermal and, to a lesser extent, chemical activation of TRPV1 provides sufficient therapeutic benefits. Such conditions are likely to entail abnormal thermal stimulation (e.g.
, due to the perfusion of the skin with the warm blood redirected from the body core, as seen in hot flushes) or thermal hyperalgesia (which is prominent in burns, frostbites, and peripheral neuropathies). Involvement of TRPV1 channels in some of these conditions has been demonstrated (Walker et al., 2003
; Bolcskei et al., 2005
). The significance of the present work, however, is limited to identifying the pharmacological profile of hyperthermia-free TRPV1 antagonists. For which conditions to use them is a subject of future research.