Native substance P with its C-terminal amide, calculated with the residue masses and pKa set used on this site.
"Preparation P"
In 1931 Ulf von Euler and John Gaddum, working in London, found that extracts of horse brain and intestine contained something that lowered blood pressure and contracted the isolated gut, and that was not acetylcholine. They dried the active material into a stable powder and labelled it simply "P", for preparation or powder. The name stuck. Von Euler later received a Nobel Prize for other work, on noradrenaline.
The structure of substance P remained unknown for four decades. In 1971 Michael Chang and Susan Leeman, who had rediscovered it in hypothalamic extracts as a potent stimulator of salivation, purified it and determined its sequence: eleven residues ending in a C-terminal methionine amide. It was synthesised shortly afterwards, and the synthetic peptide matched the natural one.
Structure
Substance P is an undecapeptide, RPKPQQFFGLM-NH₂. Its two halves have distinct roles. The N-terminal part, rich in basic residues and prolines, carries the positive charge. The C-terminal part, FFGLM-NH₂, is the region recognised by its receptors and is shared with the whole tachykinin family.
Like melittin, substance P has no acidic residues, and with its C-terminus amidated it has no acidic groups at all. Its ionisable groups are the N-terminal amine, Arg1 and Lys3, so it carries a net positive charge of about +3 at physiological pH and never becomes neutral at any pH. The isoelectric point guide uses it as the example of a peptide that has no true pI. Entered as a free acid, which is what the pI calculator assumes, it appears to have a pI of about 11.5; the difference comes entirely from the amide.
In water, substance P is largely flexible and unstructured. When bound to membranes or to its receptor, the C-terminal region adopts a helical conformation, the kind of conditional folding described in the guide to peptide secondary structure.
The tachykinin family
Substance P is the founding member of the tachykinins, a family of peptides named for their ability to contract smooth muscle quickly, in contrast to the slow-acting bradykinin. All share the C-terminal motif Phe-X-Gly-Leu-Met-NH₂, where X is an aromatic or branched aliphatic residue.
| Peptide | Source | Preferred receptor |
|---|---|---|
| Substance P | Mammals | NK1 |
| Neurokinin A | Mammals | NK2 |
| Neurokinin B | Mammals | NK3 |
| Eledoisin | Salivary glands of an octopus (Eledone) | — |
| Physalaemin | Skin of a South American frog (Physalaemus) | — |
| Kassinin | Skin of an African frog (Kassina) | — |
The non-mammalian tachykinins were among many peptides discovered by the Italian pharmacologist Vittorio Erspamer, who systematically screened amphibian skin and other animal sources from the 1940s onwards. His work showed that frog skin contains close relatives of many mammalian hormones and neuropeptides, and it often identified the non-mammalian relative before the mammalian one.
Substance P and neurokinin A are encoded on the same gene, the preprotachykinin A gene, and alternative splicing of the RNA determines which peptides a cell produces. Both are processed and amidated from precursors in the way described in the guide to post-translational modifications.
What substance P does
Substance P acts mainly through the neurokinin-1 (NK1) receptor, a G protein-coupled receptor found in the nervous system, on blood vessels, on immune cells and in the gut. Its best-studied roles are:
- Pain transmission. It is released, together with glutamate, from the central endings of sensory nerve fibres in the spinal cord in response to painful stimuli, and contributes to the transmission and amplification of pain signals.
- Neurogenic inflammation. Released from the peripheral endings of the same nerves, it dilates blood vessels, increases their permeability, and activates mast cells and immune cells. This produces the redness and swelling around an injury.
- Vomiting reflex. NK1 receptors in the brainstem regions that control vomiting are central to the emetic response.
- Gut motility and secretion, the smooth muscle effect that first revealed it.
NK1 receptor antagonists
The role of substance P in pain made the NK1 receptor an intense drug target from the 1980s. Several pharmaceutical companies developed potent, selective, non-peptide NK1 antagonists. In clinical trials, however, they proved disappointing as painkillers, and a later effort to develop them as antidepressants, after promising early results, also failed in larger trials. The gap between animal and human results in pain research is often illustrated with this example.
The same compounds turned out to be effective against the nausea and vomiting caused by some cancer chemotherapies, especially the delayed phase that other drugs controlled poorly. Aprepitant, approved in 2003, was the first NK1 antagonist to reach the clinic, and several others have followed. The story is a case study in how understanding a peptide's biology, even imperfectly, can lead to useful drugs by an unexpected route.
Stability and degradation
In the body, substance P is broken down within minutes, mainly by neprilysin and angiotensin-converting enzyme, which cleave within its C-terminal region, and by other peptidases that trim its N-terminus. The link to ACE is shared with bradykinin, as explained in the profile of angiotensins and bradykinin.
In the laboratory, the C-terminal methionine is its main weakness. Methionine oxidises readily to the sulfoxide, adding 15.995 Da, and oxidation of this residue strongly reduces activity because it lies within the receptor-binding motif. Substance P solutions should therefore be protected from air and light, stored frozen in aliquots, and checked by mass spectrometry for a +16 Da peak if activity seems low. The stability and storage guide covers the general precautions. Being strongly cationic and moderately hydrophobic, substance P also adsorbs to glass and some plastics at low concentrations.
Substance P with the tools on this site
| Property | Free acid (calculator input) | Native amide |
|---|---|---|
| Average MW | 1348.62 | 1347.64 |
| Monoisotopic mass | 1347.7121 | 1346.7281 |
| Oxidised (Met sulfoxide), monoisotopic | 1363.7070 | 1362.7230 |
| Isoelectric point | about 11.5 | None; positive at all pH values |
| Net charge at pH 7.4 | about +1.8 | about +2.8 |
Substance P is also a convenient example for mass spectrometry. Its basic N-terminal residues hold protons, so in electrospray it appears prominently as the doubly charged ion [M+2H]²⁺ at m/z 674.37, and its fragmentation is dominated by ions containing the N-terminal arginine. Its prolines produce enhanced cleavage, the "proline effect" described in the mass spectrometry guide.
Frequently asked questions
What does the "P" stand for?
Nothing more specific than "preparation" or "powder". Von Euler and Gaddum used it as a laboratory label for their dried extract, and the name was never replaced.
Is substance P a neurotransmitter or a hormone?
Mainly a neuropeptide: it is released by neurons and acts locally, often alongside a classical neurotransmitter such as glutamate. It also acts on immune and other cells near where it is released.
Why did NK1 antagonists fail as painkillers?
Several explanations have been proposed: redundancy with other pain pathways, differences between rodent and human NK1 receptors and pain processing, and the limited predictive value of animal pain models. No single explanation is universally accepted.
Why is the C-terminal amide so important?
The amidated C-terminus is part of the receptor-binding motif shared by all tachykinins. The free acid form is far less active, as is typical for amidated neuropeptides.
References
- von Euler US, Gaddum JH (1931) An unidentified depressor substance in certain tissue extracts. Journal of Physiology 72:74–87.
- Chang MM, Leeman SE, Niall HD (1971) Amino-acid sequence of substance P. Nature New Biology 232:86–87.
- Hökfelt T, Pernow B, Wahren J (2001) Substance P: a pioneer amongst neuropeptides. Journal of Internal Medicine 249:27–40.
- Hill R (2000) NK1 (substance P) receptor antagonists – why are they not analgesic in humans? Trends in Pharmacological Sciences 21:244–246.