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Cowhage is routinely used in human studies of histamine-independent itch. As such, it is of interest to understand its mechanism of action. The active component of cowhage is a cysteine protease called mucunain. As serine proteases can activate members of the protease-activated receptor (PAR) family to induce itch and pain, the mechanism of action of mucunain and cathepsin S, a human cysteine protease, was likewise thought to be through the activation of PARs. The demonstration that cathepsin S activates members of the Mas-related G-protein coupled receptor (Mrgpr) family to induce itch led us to evaluate the activity of mucunain on these receptors. We find that mucunain activates the human receptors MRPGRX1 and MRGPRX2 and induces degranulation of human mast cells. These findings indicate that Mrgprs may be involved in itch induced by cowhage.
Cowhage is the term applied to the itch-inducing spicules or trichomes that cover the seedpods of the tropical plant Mucuna pruriens. Botany purists often prefer the term Stizolobium pruriens. There are numerous terms, based on the local customs and language, equivalent to cowhage. Itching powder is one of the colloquial terms in the English language that refers to the spicules. Cowhage can also refer to the active component in the spicules, a protease, also known as mucunain.
Cowhage and histamine are the two most widely used substances employed to measure itch in human psychophysical studies. Cowhage activates mechanically sensitive sensory nerve fibers, whereas histamine activates mechanically insensitive (
). The majority of itches encountered in the clinic have a limited response to antihistamines, highlighting the importance of histamine-independent pathways. The molecular mechanism of cowhage-induced itch is thus of interest and importance with respect to understanding itch and the development of therapeutics.
In the mid-1950s, proteins, not genes, were the rage in biomedical research. Proteases, which are proteins with the capacity to cut molecules, were part of this phenomenon. Consistent with the times, it was suggested in 1955 that a protease may be the active pruritogen in cowhage, and it was given the name mucunain (
). Heat-inactivated spicules reconstituted with the plant protease induced itch in humans akin to native cowhage spicules. In addition, an irreversible inhibitor of cysteine proteases, E64, blocked the sensations induced both by native cowhage and the spicules reconstituted with mucunain (
The key to solving the problem of cowhage and cathepsin S-induced itch, and potentially conditions associated with histamine-independent itch, is a family of receptors known as Mas-related G-protein-coupled receptors or Mrgprs. Mrgprs were identified in 2001 and their role in histamine-independent itch is now clear (
). Here we report that mucunain activates human MRGPRX1 and MRGPRX2 (Figure 1).
The mechanism of cowhage-induced itch may now be explained as follows: MRGPRX1 is expressed on human dorsal root ganglion neurons. Chloroquine and BAM8-22, in addition to cowhage, activate this receptor to induce histamine-independent itch (
) allowing for cowhage to induce itch by multiple mechanisms. We acknowledge as a possible limitation that MRGPRX2 expression on neurons could result from mast cell contamination. We are also aware of the report in which transcriptional profiling from human dorsal root ganglion neurons did not identify MRGPRX2 (
), but these data were generated without amplification and do not take into account the possibility that pruritogenic or inflammatory stimuli could contribute to expression.
We propose that although cowhage primarily activates the histamine-independent pathway, it can also induce the release of histamine from mast cells. This component of cowhage-induced itch was not previously appreciated. A question then arises: If mucunain induces mast cell degranulation, why is cowhage only sometimes associated with the typical wheal and flare response of histamine injection (
)? The likely answer is that the intraepidermal nature of skin penetration by cowhage spicules combined with the limited amount of mucunain that spicules can carry and deliver is consistent with mast cells not being activated on a regular basis. In contrast, intradermal administration of histamine would allow its diffusion to reach mast cells leading to their activation. Indeed, studies with histamine at concentrations equipotent to native cowhage spicules produce sensory qualities of similar magnitude and duration, and a cowhage spicule reconstituted by histamine is capable of inducing itch and nociceptive sensations in the absence of a visible flare (
). This explanation is consistent with a study performed with plant proteases on 75 healthy subjects from the 1950s in which a variable degree of erythema and wheal formation was noted at the site of intradermal injection (
In summary, we propose that MRGPRX1 and MRGPRX2 contribute to cowhage-induced itch. These receptors represent potential therapeutic targets for histamine-independent itch.
Conflict of Interest
The authors state no conflict of interest.
EA is the recipient of a grant from the National Eczema Association. Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, R01AR057744 and R21AR067399 to EAL. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The role of proteolytic enzymes in the production of pruritus in man.