Faculty Bibliography

1. Proteases regulate cells by cleaving proteinase-activated receptors (PARs). Thrombin and trypsin cleave PAR-1 and PAR-2 on neurons and astrocytes of the brain to regulate morphology, growth and survival. We hypothesized that thrombin and mast cell tryptase, which are generated and released during trauma and inflammation, regulate enteric neurons by cleaving PAR-1 and PAR-2. 2. We detected immunoreactive PAR-1 and PAR-2 in > 60 % of neurons from the myenteric plexus of guinea-pig small intestine in primary culture. A large proportion of neurons that expressed substance P, vasoactive intestinal peptide or nitric oxide synthase also expressed PAR-1 and PAR-2. We confirmed expression of PAR-1 and PAR-2 in the myenteric plexus by RT-PCR using primers based on sequences of cloned guinea-pig receptors. 3. Thrombin, trypsin, tryptase, a filtrate from degranulated mast cells, and peptides corresponding to the tethered ligand domains of PAR-1 and PAR-2 increased [Ca2+]i in > 50 % of cultured myenteric neurons. Approximately 60 % of neurons that responded to PAR-1 agonists responded to PAR-2 agonists, and > 90 % of PAR-1 and PAR-2 responsive neurons responded to ATP. 4. These results indicate that a large proportion of myenteric neurons that express excitatory and inhibitory neurotransmitters and purinoceptors also express PAR-1 and PAR-2. Thrombin and tryptase may excite myenteric neurons during trauma and inflammation when prothrombin is activated and mast cells degranulate. This novel action of serine proteases probably contributes to abnormal neurotransmission and motility in the inflamed intestine.

Agonist-induced redistribution of G-protein-coupled receptors (GPCRs) and beta-arrestins determines the subsequent cellular responsiveness to agonists and is important for signal transduction. We examined substance P (SP)-induced trafficking of beta-arrestin1 and the neurokinin-1 receptor (NK1R) in KNRK cells in real time using green fluorescent protein. Green fluorescent protein did not alter function or localization of the NK1R or beta-arrestin1. SP induced (a) striking and rapid (<1 min) translocation of beta-arrestin1 from the cytosol to the plasma membrane, which preceded NK1R endocytosis; (b) redistribution of the NK1R and beta-arrestin1 into the same endosomes containing SP and the transferrin receptor (2-10 min); (c) prolonged colocalization of the NK1R and beta-arrestin1 in endosomes (>60 min); (d) gradual resumption of the steady state distribution of the NK1R at the plasma membrane and beta-arrestin1 in the cytosol (4-6 h). SP stimulated a similar redistribution of immunoreactive beta-arrestin1 and beta-arrestin2. In contrast, SP did not affect Galphaq/11 distribution, which remained at the plasma membrane. Expression of the dominant negative beta-arrestin319-418 inhibited SP-induced endocytosis of the NK1R. Thus, SP induces rapid translocation of beta-arrestins to the plasma membrane, where they participate in NK1R endocytosis. beta-Arrestins colocalize with the NK1R in endosomes until the NK1R recycles and beta-arrestins return to the cytosol.

Cutaneous sensory nerves mediate inflammation and wound healing by the release of neuropeptides such as substance P. Neutral endopeptidase is a cell surface enzyme that degrades substance P and thereby terminates its biologic actions. The distribution of neutral endopeptidase in normal skin and wounded human skin, however, has not been examined. The objectives of this study were to evaluate neutral endopeptidase expression in wounded and unwounded skin as well as in cells derived from human skin. Neutral endopeptidase was strikingly localized in normal skin by immunohistochemistry to keratinocytes of the epidermal basal layer, to hair follicles, eccrine and sebaceous glands as well as to endothelium of blood vessels and to large nerves. Standard incisional human wounds were studied at several time points between 1 h and 28 d after wounding. Staining for neutral endopeptidase was noted in the wound bed 6 h after wounding. In contrast to normal skin, staining of all the epidermal cell layers was noted in the migrating tongue of epithelium in l d wounds. Similar full-thickness staining was noted in 3 d and 7 d wounds in all layers of the new wound epithelium and in a "transition epithelium" near the wound edge. By 28 d post wounding neutral endopeptidase staining again was detected only in the basal layer of the epidermis. Neutral endopeptidase mRNA was detected in normal skin and wounds as well as cultured keratinocytes, fibroblasts and endothelial cells. Neutral endopeptidase enzymatic bioactivity was demonstrated in cultured keratinocytes. While it is known that several metalloproteinases important to tissue repair are produced by keratinocytes, this is the first evidence that keratinocytes produce neutral endopeptidase. Neutral endopeptidase may terminate the proinflammatory and mitogenic actions of neuropeptides in normal skin and wounds.

The multiple effects of opiate alkaloids, important therapeutic drugs used for pain control, are mediated by the neuronal miro-opioid receptor. Among the side effects of these drugs is a profound impairment of gastrointestinal transit. Endomorphins are opioid peptides recently isolated from the nervous system, which have high affinity and selectivity for micro-opioid receptors. Since the miro-opioid receptor undergoes ligand-induced receptor endocytosis in an agonist-dependent manner, we compared the ability of endomorphin-1, endomorphin-2 and the micro-opioid receptor peptide agonist, [D-Ala2,MePhe4,Gly-ol5]-enkephalin (DAMGO), to induce receptor endocytosis in cells transfected with epitope-tagged micro-opioid receptor complementary DNA, and in myenteric neurons of the guinea-pig ileum, which naturally express this receptor. Immunohistochemistry with antibodies to the FLAG epitope or to the native receptor showed that the micro-opioid receptor was mainly located at the plasma membrane of unstimulated cells. Endomorphins and DAMGO induced micro-opioid receptor endocytosis into early endosomes, a process that was inhibited by naloxone. Quantification of surface receptors by flow cytometry indicated that endomorphins' and DAMGO stimulated endocytosis with similar time-course and potency. They inhibited with similar potency electrically induced cholinergic contractions in the longitudinal muscle-myenteric plexus preparation through an action antagonized by naloxone. The apparent affinity estimate of naloxone (pA2 approximately 8.4) is consistent with antagonism at the micro-opioid receptor in myenteric neurons. These results indicate that endomorphins directly activate the micro-opioid receptor in neurons, thus supporting the hypothesis that they are ligands mediating opioid actions in the nervous system. Endomorphin-induced micro-opioid receptor activation can be visualized by receptor endocytosis.

Sensory nerves in skin are capable of releasing multiple neuropeptides, which modulate inflammatory responses by activating specific cutaneous target cells. Extravasation of particular subsets of leukocytes depends upon the regulated expression of cellular adhesion molecules such as VCAM-1 on microvascular endothelial cells. We examined the direct effect of cutaneous neuropeptides on the expression and function of human dermal microvascular endothelial cell (HDMEC) VCAM-1. A significant increase in VCAM-1 immunostaining of microvascular endothelium was observed in vivo following capsaicin application to human skin. Multiple cutaneous sensory C-fiber-released neuropeptides were evaluated for their ability to induce VCAM-1 cell surface expression on HDMEC. Only substance P (SP) was found to be capable of inducing HDMEC VCAM-1 expression. This SP-mediated VCAM-1 induction appeared to be a direct effect that did not require the release of other HDMEC-derived soluble factors. Increased HDMEC VCAM-1 mRNA expression was detected 1 h after the addition of SP, with peak mRNA increase at 6-9 h postinduction. FACS studies demonstrated a 6.5-fold increase in endothelial cell surface VCAM-1 expression detectable 16 h after addition of SP, which was specifically blocked by a neurokinin-1 receptor antagonist. Increased VCAM-1 cell surface expression on SP-treated HDMEC resulted in a 4-fold increase in the functional binding of 51Cr-labeled MOLT-4 T cells. These data indicate that SP is capable of directly and specifically up-regulating functional endothelial VCAM-1 expression and thus may play a key role in modulating certain inflammatory responses in the skin.

Inhibitory amino acids have antinociceptive actions in the spinal cord that may involve inhibition of neurotransmitter release from primary afferents. Rat spinal cord slices with dorsal roots were used to study the effect of GABA and glycine on substance P release, assessed by the internalization of neurokinin 1 receptors. After electrical stimulation of the dorsal root at 100 Hz, about half of neurokinin 1 receptor-immunoreactive neurons in laminae I-IIo showed internalization. This internalization was inhibited by GABA (100 microM) and the GABA(B) agonist R-baclofen (10 microM), but not by the GABA(A) agonist muscimol (20 microM) or glycine (100 microM). The GABA(B) antagonist 2-hydroxysaclofen (100 microM) reversed the inhibitory effect of GABA, but not the GABA(A) antagonist bicuculline (100 microM). These findings demonstrate that GABA(B) receptors, but not GABA(A) or glycine receptors, inhibit substance P release induced by dorsal root stimulation. In contrast, R-baclofen did not inhibit the internalization produced by NMDA (100 microM), indicating that the stimulatory effect of NMDA receptors on substance P release is able to surmount the inhibitory effect of GABA(B) receptors. In the presence of the GABA(B) antagonist 2-hydroxysaclofen (100 microM), but not in its absence, stimulation of the dorsal root at 1 or 10 Hz was able to elicit internalization, which was not inhibited by the NMDA receptor antagonist AP-5 (50 microM) or the channel blocker MK-801 (10 microM). Therefore, inhibition of substance P release by GABA(B) receptors is tonic, and in its absence SP release no longer requires NMDA receptor activation.

1. This study sought to determine whether neurogenic inflammation occurs in the airways by examining the effects of capsaicin or substance P on microvascular plasma leakage in the trachea and lungs of male pathogen-free C57BL/6 mice. 2. Single bolus intravenous injections of capsaicin (0.5 and 1 micromol kg(-1), i.v.) or substance P (1, 10 and 37 nmol kg(-10, i.v.) failed to induce significant leakage in the trachea, assessed as extravasation of Evans blue dye, but did induce leakage in the urinary bladder and skin. 3. Pretreatment with captopril (2.5 mg kg(-1), i.v.), a selective inhibitor of angiotensin converting enzyme (ACE), either alone or in combination with phosphoramidon (2.5 mg kg(-1), i.v.), a selective inhibitor of neutral endopeptidase (NEP), increased baseline leakage of Evans blue in the absence of any exogenous inflammatory mediator. The increase was reversed by the bradykinin B2 receptor antagonist Hoe 140 (0.1 mg kg(-1), i.v.). 4. After pretreatment with phosphoramidon and captopril, capsaicin increased the Evans blue leakage above the baseline in the trachea, but not in the lung. This increase was reversed by the tachykinin (NK1) receptor antagonist SR 140333 (0.7 mg kg(-1), i.v.), but not by the NK2 receptor antagonist SR 48968 (1 mg kg(-1), i.v.). 5. Experiments using Monastral blue pigment as a tracer localized the leakage to postcapillary venules in the trachea and intrapulmonary bronchi, although the labelled vessels were less numerous in mice than in comparably treated rats. Blood vessels of the pulmonary circulation were not labelled. 6. We conclude that neurogenic inflammation can occur in airways of pathogen-free mice, but only after the inhibition of enzymes that normally degrade inflammatory peptides. Neurogenic inflammation does not involve the pulmonary microvasculature.

Proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved by trypsin within the NH2-terminus, exposing a tethered ligand that binds and activates the receptor. We examined the secretory effects of trypsin, mediated through PAR-2, on well-differentiated nontransformed dog pancreatic duct epithelial cells (PDEC). Trypsin and activating peptide (AP or SLIGRL-NH2, corresponding to the PAR-2 tethered ligand) stimulated both an 125I- efflux inhibited by Ca2+-activated Cl- channel inhibitors and a 86Rb+ efflux inhibited by a Ca2+-activated K+ channel inhibitor. The reverse peptide (LRGILS-NH2) and inhibited trypsin were inactive. Thrombin had no effect, suggesting absence of PAR-1, PAR-3, or PAR-4. In Ussing chambers, trypsin and AP stimulated a short-circuit current from the basolateral, but not apical, surface of PDEC monolayers. In monolayers permeabilized basolaterally or apically with nystatin, AP activated apical Cl- and basolateral K+ conductances. PAR-2 agonists increased [Ca2+]i in PDEC, and the calcium chelator BAPTA inhibited the secretory effects of AP. PAR-2 expression on dog pancreatic ducts and PDEC was verified by immunofluorescence. Thus, trypsin interacts with basolateral PAR-2 to increase [Ca2+]i and activate ion channels in PDEC. In pancreatitis, when trypsinogen is prematurely activated, PAR-2-mediated ductal secretion may promote clearance of toxins and debris.