Critical evaluation of the expression of gastrin-releasing peptide in dorsal root ganglia and spinal cord

There are substantial disagreements about the expression of gastrin-releasing peptide (GRP) in sensory neurons and whether GRP antibody cross-reacts with substance P (SP). These concerns necessitate a critical revaluation of GRP expression using additional approaches. Here, we show that a widely used GRP antibody specifically recognizes GRP but not SP. In the spinal cord of mice lacking SP (Tac1 KO), the expression of not only GRP but also other peptides, notably neuropeptide Y (NPY), is significantly diminished. We detected Grp mRNA in dorsal root ganglias using reverse transcription polymerase chain reaction, in situ hybridization and RNA-seq. We demonstrated that Grp mRNA and protein are upregulated in dorsal root ganglias, but not in the spinal cord, of mice with chronic itch. Few GRP(+) immunostaining signals were detected in spinal sections following dorsal rhizotomy and GRP(+) cell bodies were not detected in dissociated dorsal horn neurons. Ultrastructural analysis further shows that substantially more GRPergic fibers form synaptic contacts with gastrin releasing peptide receptor-positive (GRPR(+)) neurons than SPergic fibers. Our comprehensive study demonstrates that a majority of GRPergic fibers are of primary afferent origin. A number of factors such as low copy number of Grp transcripts, small percentage of cells expressing Grp, and the use of an eGFP GENSAT transgenic as a surrogate for GRP protein have contributed to the controversy. Optimization of experimental procedures facilitates the specific detection of GRP expression in dorsal root ganglia neurons

Spinal k-Opioid Receptor Activation Attenuates Itch by Inhibiting Gastrin-releasing Peptide Receptor Function

Chronic itch is a major unmet problem that cannot be treated with anti-histamines. Itch information is relayed from the skin to the brain via the spinal cord. Previous studies show that systemic activation of classically Gαi-coupled -opioid receptor (KOR) inhibits histaminergic and non-histaminergic itch. Nalfurafine, a KOR agonist, has been used in clinical trials to treat pruritus (itch), and has been approved to treat uremic itch in Japan. However, the underlying mechanisms are unknown. Gastrin-releasing peptide (GRP), a neurotransmitter released from sensory neurons and its receptor, gastrin-releasing peptide receptor (GRPR), an itch-specific receptor expressed in the dorsal horn of the spinal cord, are primarily required for mediating non-histaminergic itch. GRPR is a Gαq-coupled receptor that is also crucial for the development of chronic itch in mouse models. Here, we hypothesize that spinal KOR activation inhibits histamine-independent itch by blocking GRPR function. We found that spinal KOR activation by an exogenous KOR agonist U-50,488 attenuated histamine-independent itch and GRP-induced calcium responses of GRPR neurons via a Gαi-independent pathway and these effects can be mimicked by protein kinase C (PKC) activation in a Ca2+-independent manner. Using isoform specific siRNA knockdown approaches, we identified PKCδ as the isoform that mediates KOR activation induced itch inhibition. Conversely, blocking PKC activation abolishes KOR activation induced inhibition of itch signaling. KOR is co-expressed with GRPR in the dorsal horn of the spinal cord, and KOR heteromerizes with GRPR in human embryonic kidney (HEK293) cells. KOR activation induced a rapid and robust GRP-independent cross-phosphorylation of GRPR in HEK293 cells, which could be blocked by PKC inhibition. Interestingly, U-50,488 blocked GRP- but not AMPA-induced inward currents in GRPR neurons, suggesting that glutamatergic transmission is not involved in KOR-mediated inhibition of GRPR function. Mice lacking KOR or dynorphin, the endogenous agonist for KOR, display normal acute and chronic itch behavior, indicating that the endogenous dynorphin/KOR system is not involved in the inhibition of itch. Taken together, our studies suggest that spinal KOR inhibits itch by blocking the function of GRPR via Gαi- and Ca2+-independent PKC activation and phosphorylation

Facilitation of TRPV4 by TRPV1 is required for itch transmission in some sensory neuron populations

The transient receptor potential channels (TRPs) respond to chemical irritants and temperature. TRPV1 responds to the itch-inducing endogenous signal histamine, and TRPA1 responds to the itch-inducing chemical chloroquine. We showed that, in sensory neurons, TRPV4 is important for both chloroquine- and histamine-induced itch and that TRPV1 has a role in chloroquine-induced itch. Chloroquine-induced scratching was reduced in mice in which TRPV1 was knocked down or pharmacologically inhibited. Both TRPV4 and TRPV1 were present in some sensory neurons. Pharmacological blockade of either TRPV4 or TRPV1 significantly attenuated the Ca(2+) response of sensory neurons exposed to histamine or chloroquine. Knockout of Trpv1 impaired Ca(2+) responses and reduced scratching behavior evoked by a TRPV4 agonist, whereas knockout of Trpv4 did not alter TRPV1-mediated capsaicin responses. Electrophysiological analysis of human embryonic kidney (HEK) 293 cells coexpressing TRPV4 and TRPV1 revealed that the presence of both channels enhanced the activation kinetics of TRPV4 but not of TRPV1. Biochemical and biophysical studies suggested a close proximity between TRPV4 and TRPV1 in dorsal root ganglion neurons and in cultured cells. Thus, our studies identified TRPV4 as a channel that contributes to both histamine- and chloroquine-induced itch and indicated that the function of TRPV4 in itch signaling involves TRPV1-mediated facilitation. TRP facilitation through the formation of heteromeric complexes could be a prevalent mechanism by which the vast array of somatosensory information is encoded in sensory neurons

In situ

Non-enzymatic glycation of extracellular matrix with (U-13C5)-d-ribose-5-phosphate (R5P), enables in situ 2D ssNMR identification of many deleterious protein modifications and crosslinks, including previously unreported oxalamido and hemiaminal (CH3-CH(OH)NHR) substructures. Changes in charged residue proportions and distribution may be as important as crosslinking in provoking and understanding harmful tissue changes.</p