Nerol

A Wikipedia nerol page claims that nerol was named after neroli essential oil. Nerol is one of many terpenes in AG Industries neroli oil.

This is another ectopic olfactory receptor saga. This time the receptor, OR2w3 is expressed in airway smooth muscle and the thyroid. OR2w3 is a light weight in terms of G protein coupled receptors. OR2w3 has almost no N-terminus extracellular domain and almost no C-terminus intracellular domain. OR2w3 weighs in at a mere 35 kDa. The authors of the first publication were looking for an asthma treatment. The second group was looking for a thyroid cancer treatment. The first group found that nerol decreased airway smooth muscle cell stiffness, a surrogate for contraction. [1] The second group examined three different thyroid cancer cell lines. They found that nerol inhibited invasion into a collagen matrix and migration without affecting proliferation. [2] This image was derived from ProteinAtlas.org. The features organs are starred. .

OR2w3 is an oddball in the family of G protein coupled receptors. This image was derived from UniProt.org. Amazingly, there is nothing in UniProt regarding phosphorylation sites of OR2w3.This is a story bout smooth muscle that includes the H1 histamine receptor in smooth muscle. HRH1 is also a G protein coupled receptor. UniProt lists nine phosphorylation sites and one disulfide bond. While most predicted domains of these receptors with seven cell membrane spanning helical domains are about the same, HRH1 has a considerablylarger cytoplasmic comain between the 6^th and 7^th transmembrane domains.

Nerol and airway smooth muscle relaxation, Paradox solved

Odor receptors are G protein coupled receptors in nasal epithelial cells. Their discovery in air way smooth muscle cells suggests that airway contraction and/or relaxation may be mediated by small molecules in the air. This has particular relevance to those that suffer from asthma. Previous studies had established that nerol caused calcium influx and bronchiole relaxation in human precision cut lung sections. The authors also cited work showing that inhibition of OR2W3 abolished the response to nerol in cultured ASM cells. This is in itself is a paradox. Increases in intracellular Ca²⁺ are generally associated with Ca²⁺-calmodulin binding to myosin light chain kinase, phosphorylation of the regulatory light chain of myosin light chain, and activation of the myosin head motor. Huange and coworkers used FLIPPR technology to measure changes in intracellular Ca²⁺. [1] Twenty compounds, with seven different classes of odors, were screened with human ASM cells. [1] Contraction was determined by attaching magnetic beads to the surface of the cells by coating the beads with RGD peptides. The arginine-glycine-aspartic peptide binds to integrin molecules on the cell surface. Sinusoidal magnetic forces were applied to the beads. Beads attached to relaxed cells rotate more freely than those attached to ASM that are contracting. [1]

compounds tested and results

The first row shows chemical structures. Relative fluorescence changes of an intracellular Ca²⁺ sensitive compound are shown in the second row. Changes in air way smooth muscle cell stiffness, as a surrogate for contraction, are in the third row.

• Linalool does not cause an increase in intracellular Ca²⁺, but does cause an increase in tension that then decreases to below baseline.
• Nerol causes a Ca²⁺ spike that coincides with a decrease in tension.
• Geraniol is essentially the same compound as nerol except that the first double bond is cis trans in geraniol.  The same phenomenon was observed.  
• Citronellol has only one double bond, causes no change in intracellular Ca²⁺ and only a spike increase in tension with no decrease below baseline.
• Eugenol has a different structure, but causes a Ca²⁺ spike followed by a sustained decrease in tension. 
• Histamine is a positive control.  It causes Ca²⁺ to spike followed by a sustained increase in tension.  This is what conventional wisdom says is supposed to happen when intracellular Ca²⁺ rise.

2.  Establishing a dose response

The EC₅₀ for intracellular calcium doesn’t seem that impressive at about 1mM.  Cell stiffness is a little more plausible.  The Huang and coworkers also looked at precision cut lung samples from three different donors. [1] Carbachol is an agonist of nicotinic and muscarinic acetyl choline receptors. The G protein coupling is isoform dependent. Mickry 2005 is the source of some of these cartoons.

Forskolin is a small molecule activator of adenylyl cyclase.  Carbachol without nerol causes vasoconstriction suggesting G_q signalling. Even though nerol increases intracellular Ca²⁺, it causes relaxation on top of carbachol. The paradox continues.

3.  ↑ Ca²⁺ : Is it Gα_q or a TRP family member?

This site has documented Transient Receptor Potential  Ca²⁺ channel family members are often gated by terpenes.  OR2w3 may signal via Gα_q to increase intracellular Ca²⁺.  Huang and coworkers used antagonists specific to the individual family TRP family members.   

Nerol does not appear to be increasing intracellular Ca²⁺ via these three TRP family members.

4.  OR2w3 is expressed in the ASM cell line and in normal human tissue

The double green bands in Fig4C are highly suggestive of splice variants, but we know so very little about OR2w3.  UniProt makes no mention of a signal peptide but does present evidence for N-terminal glycosylation based on sequence homology.  Phosphorylation may alter the migration of a protein in an SDS PAGE gel Phosphorylation often results in a large conformational change that results in changes in migration in SDS PAGE gels..  The supplemental data show a little bit less of the double bands in 10% reducing agent as opposed to 5%.  More reducing agent causes OR2w3 to run slower suggestive of a more extended structure    Our comparison G protein receptor HRH1 has a disulfide bond according to UniProt.

5. OR2w3 does not fire through Gα_s or Gα_i

This is a composite figure that uses parts of supplemental figures 1 and 2. [1] The silencing RNA control shControl is just junk nonsense RNA that should not change the OR2w3 expression. Note that silencing OR2w3 does not affect other olfactory receptors that might also be activated by nerol. [1]

The exciting thing about the 5C results in panel 5C is that we are now getting into the realm of achievable concentrations in a human or a rat by going to the surrogate of contraction: cell stiffness. In the 62.5 μM panel the 240-300 second segment of the graph was enlarged to show the different between the wild type and knockdown ASM cells.

6  What’s the target of OR2w3?

Figure 6A looks at the possibility of Gβν coupled to the large (Big) conductance Ca²⁺ activated K⁺ channel and to IP3 producing Phospholipase Cβ that is activated by Gαq. IP3 evokes release of Ca²⁺ from the sarcoplasmic reticulum. The Ca²⁺ chelator EGTA has no effect on histamine induced contraction but diminishes the nerol induced relaxation. (Panel B) Thapsigargin is an inhibitor of the sarcoplasmic, endoplasmic reticulum calcium reuptake ATPase.

Panels 6C and C show promising dose response data.

7, It’s the Ca²⁺ and voltage activated Cl^– channel

This particular voltage and Ca²⁺ channel is known by different names. TMEM16A, ANOI1 (the gene), and anoctamin are names of these chloride channels found in smooth muscle and olfactory epithelial cells. Note that the resting membrane potential is outside positive relative to the inside negative.

8. And the CFTR chloride channel too?

CFTR is widely known as the cysic fibrosis transmembrane conductance regulator. It is widely recognized as the driver of chloride secretion i the apical membranes of not only airway epithelial cells but also other secretory epithelium. Even the Wikipedia scholars seem to have overlooked the interaction of CFTR with the plasma membrane Ca2+ ATPase (PMCA) as well as SERCA of the endoplasmic reticulum of pulmonary epithelial cells. [3] Dysfunctional CFTR seems to result in incrased SERCA pump activity in the ER. [3]

But SERCA inhibitor thapsigairgin did nothing to nerol relaxation but inhibited the histamine HRH1 contraction.

And thyroid cancer too

The work of Huang and coworkers (2020) can apply to other cells that express OR2w3. OR2W3 expression in follicular and papillary thyroid tumors was just 25-30% of surrounding normal tissue. [2] This publication was primarily about a different olfactory receptor and synthetic ligand. The authors treated the cells with 500 µM nerol for the proliferation assay and250 µM nerol for migration and invasion. [2]
Proliferation was defined as incorporation of the uridine analog BrdU. Migration was defined as Invasion was defined by migration into a Matrigel matrix that required digestion of this the matrix.

Cell line	Tumor origin	OR2w3	proliferation	migration	invasion
FTF-133	follicular	~0.05	same	same	~80%↑
BPCAP	papillary	0.2	same	~15%↓	~25%↓
Nthy-ori 3-1	Metastasis	1.0	same	~30% ↓	~60%↓

CFTR associates with SERCA in airway epithelial cells. [2] Nerol inibits contraction of ASM [1] and migration of an epithelial cancer [3]. These data suggest that OR2w3 are part of a larger complex. Huang and coworkers presented cartoons describing their working model. CFTR conducatance is modulated by cAMP activated protein kinase A, yet OR2w3 does not modulate adenylyl cyclase.

Cartoons for a deeper understanding of OR2w3 vs HRH1

Many summary cartoons in Huang 2020 [1] are not being presented because they show a well spaced configuration of the Ca²⁺ and Cl^– channels whereas the cartoons in the reviews of epithelial cells [3] and muscle cells [4] show tight packing. Does not having the extra C-terminus baggage help it pack tighter with targets so as to limit diffusion of second messengers?

References

1. Huang J, Lam H, Koziol-White C, Limjunyawong N, Kim D, Kim N, Karmacharya N, Rajkumar P, Firer D, Dalesio NM, Jude J, Kurten RC, Pluznick JL, Deshpande DA, Penn RB, Liggett SB, Panettieri RA Jr, Dong X, An SS.(2020)  The odorant receptor OR2W3 on airway smooth muscle evokes bronchodilation via a cooperative chemosensory tradeoff between TMEM16A and CFTR. Proc Natl Acad Sci U S A. 2020 Nov 10;117(45):28485-28495.  PMC free article
2. Weidinger, D., Jovancevic, N., Zwanziger, D., Theurer, S., Hönes, J., Führer, D., & Hatt, H. (2021). Functional Characterization of Olfactory Receptors in the Thyroid Gland. Frontiers in physiology, 12, 676907. PMC free article
3. Philippe R, Antigny F, Buscaglia P, Norez C, Becq F, Frieden M, Mignen O. SERCA and PMCA pumps contribute to the deregulation of Ca2+ homeostasis in human CF epithelial cells. Biochim Biophys Acta. 2015 May;1853(5):892-903. free article
4. Avila-Medina, J., Mayoral-Gonzalez, I., Dominguez-Rodriguez, A., Gallardo-Castillo, I., Ribas, J., Ordoñez, A., Rosado, J. A., & Smani, T. (2018). The Complex Role of Store Operated Calcium Entry Pathways and Related Proteins in the Function of Cardiac, Skeletal and Vascular Smooth Muscle Cells. Frontiers in physiology, 9, 257. PMC free article