Faculty Bibliography

The effects of proteinase-activated receptor 2 (PAR2) agonists on the electrical properties of intact guinea pig ileal myenteric neurons were measured with intracellular microelectrodes. Approximately 52% of AH neurons and 41% of S neurons responded to pressure ejection of SLIGRL-NH(2) or trypsin with a prolonged depolarization that was often accompanied by increased excitability. When added to the bathing solution, trypsin caused a concentration-dependent depolarization of responding neurons with an estimated EC(50) value of 87 nM. Collectively, these novel observations indicate that PAR2 excites a proportion of myenteric neurons, which may contribute to dysmotility during intestinal inflammation.

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 (PAR2) to induce alterations in contraction of airway smooth muscle that have been implicated in asthma in experimental animals. Although tryptase inhibitors are under development for treatment of asthma, little is known about the localization and function of PAR2 in human airways. We detected PAR2 expression in primary cultures of human airway smooth muscle cells using reverse transcriptase/polymerase chain reaction (RT-PCR) and immunofluorescence. The PAR2 agonists trypsin, tryptase, and an activating peptide (SLIGKV-NH2) stimulated calcium mobilization in these cells. PAR2 agonists strongly desensitized responses to a second challenge of trypsin and SLIGKV-NH2, but not to thrombin, indicating that they activate a receptor distinct from the thrombin receptors. Immunoreactive PAR2 was detected in smooth muscle, epithelium, glands, and endothelium of human bronchi. Trypsin, SLIGKV-NH2, and tryptase stimulated contraction of isolated human bronchi. Contraction was increased by removal of the epithelium and diminished by indomethacin. Thus, PAR2 is expressed by human bronchial smooth muscle where its activation mobilizes intracellular Ca2+ and induces contraction. These results are consistent with the hypothesis that PAR2 agonists, including tryptase, induce bronchoconstriction of human airway by stimulating smooth muscle contraction. PAR2 antagonists may be useful drugs to prevent bronchoconstriction.

Toxin A (TxA) of Clostridium difficile induces acute inflammation of the intestine initiated by release of substance P (SP) and activation of the neurokinin-1 receptor. However, the mechanisms that terminate this response are unknown. We determined whether the SP-degrading enzyme neutral endopeptidase (NEP, EC 3.4.24.11) terminates TxA-induced enteritis. We used both genetic deletion and pharmacological inhibition of NEP to test this hypothesis. In wild-type mice, instillation of TxA (0.5-5 microg) into ileal loops for 3 h dose dependently increased ileal fluid secretion, stimulated granulocyte transmigration determined by myeloperoxidase activity, and caused histological damage characterized by depletion of enterocytes, edema, and neutrophil accumulation. Deletion of NEP reduced the threshold secretory and inflammatory dose of TxA and exacerbated the inflammatory responses by more than twofold. This exacerbated inflammation was prevented by pretreatment with recombinant NEP. Conversely, pretreatment of wild-type mice with the NEP inhibitor phosphoramidon exacerbated enteritis. Thus NEP terminates enteritis induced by C. difficile TxA, underlying the importance of SP degradation in limiting neurogenic inflammation.

Thrombin, generated in the circulation during injury, cleaves proteinase-activated receptor 1 (PAR1) to stimulate plasma extravasation and granulocyte infiltration. However, the mechanism of thrombin-induced inflammation in intact tissues is unknown. We hypothesized that thrombin cleaves PAR1 on sensory nerves to release substance P (SP), which interacts with the neurokinin 1 receptor (NK1R) on endothelial cells to cause plasma extravasation. PAR1 was detected in small diameter neurons known to contain SP in rat dorsal root ganglia by immunohistochemistry and in situ hybridization. Thrombin and the PAR1 agonist TFLLR-NH(2) (TF-NH(2)) increased [Ca(2+)](i) >50% of cultured neurons (EC(50)s 24 mu ml(-1) and 1.9 microM, respectively), assessed using Fura-2 AM. The PAR1 agonist completely desensitized responses to thrombin, indicating that thrombin stimulates neurons through PAR1. Injection of TF-NH(2) into the rat paw stimulated a marked and sustained oedema. An NK1R antagonist and ablation of sensory nerves with capsaicin inhibited oedema by 44% at 1 h and completely by 5 h. In wild-type but not PAR1(-/-) mice, TF-NH(2) stimulated Evans blue extravasation in the bladder, oesophagus, stomach, intestine and pancreas by 2 - 8 fold. Extravasation in the bladder, oesophagus and stomach was abolished by an NK1R antagonist. Thus, thrombin cleaves PAR1 on primary spinal afferent neurons to release SP, which activates the NK1R on endothelial cells to stimulate gap formation, extravasation of plasma proteins, and oedema. In intact tissues, neurogenic mechanisms are predominantly responsible for PAR1-induced oedema.

Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca(2+) mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP.

Using a combined pharmacological and gene-deletion approach, we have delineated a novel mechanism of neurokinin-1 (NK-1) receptor-dependent hyperalgesia induced by proteinase-activated receptor-2 (PAR2), a G-protein-coupled receptor expressed on nociceptive primary afferent neurons. Injections into the paw of sub-inflammatory doses of PAR2 agonists in rats and mice induced a prolonged thermal and mechanical hyperalgesia and elevated spinal Fos protein expression. This hyperalgesia was markedly diminished or absent in mice lacking the NK-1 receptor, preprotachykinin-A or PAR2 genes, or in rats treated with a centrally acting cyclooxygenase inhibitor or treated by spinal cord injection of NK-1 antagonists. Here we identify a previously unrecognized nociceptive pathway with important therapeutic implications, and our results point to a direct role for proteinases and their receptors in pain transmission.

BACKGROUND & AIMS/OBJECTIVE:N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that have an important role in long-term potentiation and memory processing in the central nervous system. The aims in this study were to determine whether NMDA receptors are expressed in the peripheral nervous system and identify their role in mediating behavioral pain responses to colonic distention in the normal gut. METHODS AND RESULTS/RESULTS:Immunohistochemical localization of the NR1 subunit showed that NMDA receptors are expressed on the cell bodies and peripheral terminals of primary afferent nerves innervating the colon. Dorsal root ganglia neurons retrogradely labeled from the colon in short-term culture responded to addition of NMDA with increased intracellular [Ca2+]. Activation of peripheral NMDA receptors in colonic tissue sections caused Ca2+-dependent release of the proinflammatory neuropeptides, calcitonin gene-related peptide and substance P. Behavioral pain responses to noxious mechanical stimulation were inhibited in a reversible, dose-dependent manner by intravenous administration of memantine, a noncompetitive antagonist of the NMDA receptor. Single fiber recordings of decentralized pelvic nerves showed that colorectal distention responsive afferent nerve activity was inhibited by memantine. CONCLUSIONS:Peripheral NMDA receptors are important in normal visceral pain transmission, and may provide a novel mechanism for development of peripheral sensitization and visceral hyperalgesia.

An understanding of the mechanisms that regulate signaling by the substance P (SP) or neurokinin 1 receptor (NK1-R) is of interest because of their role in inflammation and pain. By using activators and inhibitors of protein kinase C (PKC) and NK1-R mutations of potential PKC phosphorylation sites, we determined the role of PKC in desensitization of responses to SP. Activation of PKC abolished SP-induced Ca(2+) mobilization in cells that express wild-type NK1-R. This did not occur in cells expressing a COOH-terminally truncated NK1-R (NK1-Rdelta324), which may correspond to a naturally occurring variant, or a point mutant lacking eight potential PKC phosphorylation sites within the COOH tail (NK1-R Ser-338, Thr-339, Ser-352, Ser-387, Ser-388, Ser-390, Ser-392, Ser-394/Ala, NK1-RKC4). Compared with wild-type NK1-R, the t(1/2) of SP-induced Ca(2+) mobilization was seven- and twofold greater in cells expressing NK1-Rdelta324 and NK1-RKC4, respectively. In cells expressing wild-type NK1-R, inhibition of PKC caused a 35% increase in the t(1/2) of SP-induced Ca(2+) mobilization. Neither inhibition of PKC nor receptor mutation affected desensitization of Ca(2+) mobilization to repeated challenge with SP or SP-induced endocytosis of the NK1-R. Thus PKC regulates SP-induced Ca(2+) mobilization by full-length NK1-R and does not regulate a naturally occurring truncated variant. PKC does not mediate desensitization to repeated stimulation or endocytosis of the NK1-R.

Agonist-induced endocytosis of somatostatin receptors determines subsequent cellular responsiveness to peptide agonist and influences somatostatin receptor scintigraphy, a technique to image various tumours. We examined the internalization of sst3HSV, an epitope-tagged type 3 somatostatin receptor, in transfected rat neuroendocrine insulinoma cells. Stimulation of these cells with somatostatin induced trafficking of coexpressed enhanced green fluorescence protein/beta-arrestin1 fusion protein and sst3HSV to colocalize in the same endocytic vesicles. Coexpression of a dominant negative mutant of the arrestin fusion protein with the receptor blocked the internalization of sst3HSV. Stimulation with somatostatin also induced the transient translocation of alpha-adaptin, a component of the adaptor protein complex 2, to the plasma membrane. alpha-adaptin and clathrin colocalized with the receptor. By electron microscopy, we observed internalized sst3 in clathrin coated pits, endosomes and at the limiting membrane of multivesicular bodies, a location typical for receptors being recycled. Concordantly, we observed sst3HSV colocalized with Rab11 in a perinuclear compartment which is likely to correspond to the pericentriolar recycling endosome. Thus, agonist-induced endocytosis of sst3 depends on its interaction with beta-arrestin, involves the adaptor protein complex 2 and proceeds via clathrin coated vesicles to the recycling compartment.