Abstract
Substance P (SP) is a prototypical neuropeptide with roles in pain and inflammation. Numerous mechanisms regulate endogenous SP levels, including the differential expression of SP mRNA and the controlled secretion of SP from neurons. Proteolysis has long been suspected to regulate extracellular SP concentrations but data in support of this hypothesis is scarce. Here, we provide evidence that proteolysis controls SP levels in the spinal cord. Using peptidomics to detect and quantify endogenous SP fragments, we identify the primary SP cleavage site as the C-terminal side of the ninth residue of SP. If blocking this pathway increases SP levels, then proteolysis controls SP concentration. We performed a targeted chemical screen using spinal cord lysates as a proxy for the endogenous metabolic environment and identified GM6001 (galardin, ilomastat) as a potent inhibitor of the SP 1–9-producing activity present in the tissue. Administration of GM6001 to mice results in a greater-than-three-fold increase in the spinal cord levels of SP, which validates the hypothesis that proteolysis controls physiological SP levels.
Highlights
A member of the tachykinin family of neuropeptides, substance P (SP) is an amidated undecapeptide (Fig. 1) that is widely expressed in the central and peripheral nervous systems [1] of mammals and functions as a neurotransmitter and neuromodulator [2]
One cannot conclude from the mere presence of SP-degrading activity in SP-containing tissues that proteolysis controls SP levels because it is possible that the enzymes responsible for the observed activities do not physically contact endogenous SP in the cell or are otherwise prevented from acting on the peptide
The data indicates that the primary SP-degrading activity in spinal cord resides in the membrane fraction and that proteolysis of SP occurs through Cterminal processing, which is consistent with previous in vitro lysate experiments [32,40,41]
Summary
A member of the tachykinin family of neuropeptides, substance P (SP) is an amidated undecapeptide (Fig. 1) that is widely expressed in the central and peripheral nervous systems [1] of mammals and functions as a neurotransmitter and neuromodulator [2] It participates in a host of fundamentally and biomedically important physiological processes, including pain transmission [3,4,5], inflammation [6,7], sleep [8], learning and memory [9,10], depression and affective mood disorders [11,12,13], opioid dependence [14,15,16] and apoptosis [17,18]. Metabolic studies are not sufficient to establish proteolysis as a mechanism of SP regulation
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