Faculty Bibliography

There is increasing evidence that sensory nerves may participate in cutaneous inflammatory responses by the release of neuropeptides such as substance P (SP). We examined the direct effect of SP on human dermal microvascular endothelial cell (HDMEC) intercellular adhesion molecule 1 (ICAM-1) expression and function. Our results indicated that, although cultured HDMEC expressed mRNA for neurokinin receptors 1, 2, and 3 (NK-1R, NK-2R, and NK-3R), SP initiated a rapid increase in HDMEC intracellular Ca2+ levels, primarily by the activation of NK-1R. Immunohistochemistry studies likewise demonstrated that HDMEC predominantly expressed NK-1R. The addition of SP to HDMEC resulted in a rapid increase in cellular ICAM-1 mRNA levels, followed by a fivefold increase in ICAM-1 cell surface expression. This functionally resulted in a threefold increase in 51Cr-labeled binding of J-Y lymphoblastoid cells to HDMEC. In vivo studies demonstrated a marked increase in microvascular ICAM-1 immunostaining 24 and 48 h after application of capsaicin to the skin. These results indicate that neuropeptides such as SP are capable of directly activating HDMEC to express increased levels of functional ICAM-1 and further support the role of the cutaneous neurological system in modulating inflammatory processes in the skin.

Although proteases are traditionally viewed as degradative enzymes, characterization of a family of G protein-coupled receptors that are activated by proteolysis reveals a new role. Certain proteases function as signaling molecules that specifically regulate cells by cleaving and activating a family of proteinase-activated receptors.

Observations in reconstituted systems and transfected cells indicate that G-protein receptor kinases (GRKs) and beta-arrestins mediate desensitization and endocytosis of G-protein-coupled receptors. Little is known about receptor regulation in neurons. Therefore, we examined the effects of the neurotransmitter substance P (SP) on desensitization of the neurokinin-1 receptor (NK1-R) and on the subcellular distribution of NK1-R, Galphaq/11, GRK-2 and -3, and beta-arrestin-1 and -2 in cultured myenteric neurons. NK1-R was coexpressed with immunoreactive Galphaq/11, GRK-2 and -3, and beta-arrestin-1 and -2 in a subpopulation of neurons. SP caused 1) rapid NK1-R-mediated increase in [Ca2+]i, which was transient and desensitized to repeated stimulation; 2) internalization of the NK1-R into early endosomes containing SP; and 3) rapid and transient redistribution of beta-arrestin-1 and -2 from the cytosol to the plasma membrane, followed by a striking redistribution of beta-arrestin-1 and -2 to endosomes containing the NK1-R and SP. In SP-treated neurons Galphaq/11 remained at the plasma membrane, and GRK-2 and -3 remained in centrally located and superficial vesicles. Thus, SP induces desensitization and endocytosis of the NK1-R in neurons that may be mediated by GRK-2 and -3 and beta-arrestin-1 and -2. This regulation will determine whether NK1-R-expressing neurons participate in functionally important reflexes.

Although serine proteases are usually considered to act principally as degradative enzymes, certain proteases are signaling molecules that specifically regulate cells by cleaving and triggering members of a new family of proteinase-activated receptors (PARs). There are three members of this family, PAR-1 and PAR-3, which are receptors for thrombin, and PAR-2, a receptor for trypsin and mast cell tryptase. Proteases cleave within the extracellular NH2-terminus of their receptors to expose a new NH2-terminus. Specific residues within this tethered ligand domain interact with extracellular domains of the cleaved receptor, resulting in activation. In common with many G protein-coupled receptors, PARs couple to multiple G proteins and thereby activate many parallel mechanisms of signal transduction. PARs are expressed in multiple tissues by a wide variety of cells, where they are involved in several pathophysiological processes, including growth and development, mitogenesis, and inflammation. Because the cleaved receptor is physically coupled to its agonist, efficient mechanisms exist to terminate signaling and prevent uncontrolled stimulation. These include cleavage of the tethered ligand, receptor phosphorylation and uncoupling from G proteins, and endocytosis and lysosomal degradation of activated receptors.

The use of angiotensin-converting enzyme (ACE) has been associated with the occurrence of adverse effects, including cough and angioneurotic edema. Accumulation of kinins has been suggested to play a major role in these adverse effects of ACE inhibitor, although conclusive evidence for such a role is lacking. We investigated whether ACE inhibition increases plasma extravasation in mice (Swiss, C57Bl/6J, and J129Sv/Ev strains) via inhibition of bradykinin metabolism and stimulation of neurogenic inflammatory mechanisms. Intravenous captopril and enalapril increased the extravasation of Evans blue dye in all tissues examined (trachea, stomach, duodenum, and pancreas). This effect was evident 15 minutes after drug administration. The particulate dye Monastral blue identified the sites of captopril-induced leakage in the microvasculature. Pretreatment with the bradykinin B2 receptor antagonist Hoe 140 or with the tachykinin NK1 receptor antagonist SR 140333 inhibited captopril-evoked increase in plasma extravasation. In mice in which the gene encoding the bradykinin B2 receptor was disrupted by gene targeting, neither bradykinin nor captopril increased plasma extravasation. Pretreatment with Hoe 140 did not reduce the hypotensive response induced by captopril. The present findings suggest that ACE inhibition increases kinin levels in tissues and/or plasma. These increased kinin levels increase microvascular leakage in mouse airways and digestive tract via the release of tachykinins from terminals of primary sensory neurons. Exaggerated kinin production and the subsequent stimulation of peptide release from sensory nerves may be involved in adverse effects of ACE inhibitors.

Neuropeptides exert their diverse biological effects by interacting with G protein-coupled receptors (GPCRs). In this review we address the question, What regulates the ability of a target cell, in particular a neuron, to respond to a neuropeptide? Available evidence from studies of many GPCRs in reconstituted systems and transfected cell lines indicates that much of this regulation occurs at the level of the receptor and serves to alter the capacity of the receptor to bind ligands with high affinity and to couple to heterotrimeric G proteins. Although some of the knowledge gained from these studies is applicable to the regulation of neuropeptide receptors on neurons, at present there are far more questions than answers.

OBJECTIVE:To determine which types of kinin receptor are present in human bronchial epithelial cells we studied the capability of bradykinin to mobilize intracellular Ca2+ ([Ca2+]i) in a human bronchial epithelial cell line (16HBE cells). MATERIAL/METHODS:Human bronchial epithelial cell line transformed with an original defective simian virus 40 (SV40). TREATMENT/METHODS:Bradykinin (0.1 pM to 0.1 microM), des-Arg9 bradykinin (1 microM), des-Arg10) kallidin (1 microM), indomethacin (1 microM), phosphoramidon (1 microM), captopril (1 microM), des-Arg9-[Leu8]bradykinin (1 microM), HOE 140 (DArg-[Hyp3, Thi5, DTic , Oic8]-bradykinin) (1 microM), and NPC 16731 (DArg-[Hyp3, Thi5, DTic7, Tic8]-bradykinin) (1 microM). METHODS:The mobilization of [Ca2+]i was determined by the fura-2 method. Two sample Wilcoxon rank-sum (Mann-Whitney) test was used for statistical calculations. RESULTS:Bradykinin, but not the selective agonists for kinin B1 receptor des-Arg9 bradykinin and des-Arg10 kallidin, increased the mobilization of [Ca2+]i (EC50, 0.079+/-0.009nM) in 16HBE cells in a concentration-dependent manner. Pretreatment with the cyclooxygenase inhibitor indomethacin (1 microM) or the peptidase inhibitors, phosphoramidon (1 microM) or captopril (1 microM), did not affect the response to bradykinin. The kinin B1 receptor antagonist, des-Arg9-[Leu8]bradykinin (1 microM), was inactive. HOE 140 and NPC 16731, two selective antagonists of the kinin B2 receptor abolished the response to bradykinin (IC50 of HOE 140 and NPC 16731 were 0.52+/-0.037nM and 1.67 +/- 0.41 nM, respectively). CONCLUSIONS:The present data indicate the presence of kinin B2 receptors in the 16HBE cells.

The interaction between components of the nervous system and multiple target cells in the cutaneous immune system has been receiving increasing attention. It has been observed that certain skin diseases such as psoriasis and atopic dermatitis have a neurogenic component. Neuropeptides released by sensory nerves that innervate the skin and often contact epidermal and dermal cells can directly modulate functions of keratinocytes, Langerhans cells (LC), mast cells, dermal microvascular endothelial cells and infiltrating immune cells. Among these neuropeptides the tachykinins substance P (SP) and neurokinin A (NKA), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and somatostatin (SOM) have been reported to effectively modulate skin and immune cell functions such as cell proliferation, cytokine production or antigen presentation under physiological or pathophysiological conditions. Expression and regulation of their corresponding receptors that are expressed on a variety of skin cells as well as the presence of neuropeptide-specific peptidases such as neutral endopeptidase (NEP) or angiotensin-converting enzyme (ACE) determine the final biological response mediated by these peptides on the target cell or tissue. Likewise, skin cells like keratinocytes or fibroblasts are a source for neurotrophins such as nerve growth factor that are required not only for survival and regeneration of sensory neurons but also to control responsiveness of these neurons to external stimuli. Therefore, neuropeptides, neuropeptide receptors, neuropeptide-degrading enzymes and neurotrophins participate in a complex, interdependent network of mediators that modulate skin inflammation, wound healing and the skin immune system. This review will focus on recent studies demonstrating the role of tachykinins, CGRP, SOM and VIP and their receptors and neuropeptide-degrading enzymes in mediating neurogenic inflammation in the skin.

The excitability of spinal neurons that transmit pain is modulated by glutamate and substance P (SP). Glutamate is an excitatory neurotransmitter in the dorsal horn, and its effects are enhanced by SP acting on neurokinin 1 receptors (NK1Rs). We assessed activation of NK1Rs by studying their internalization in spinal cord slices. NK1Rs were localized in sections from the slices by using immunohistochemistry combined with fluorescence and confocal microscopy. Incubating the slices with SP induced internalization in most NK1R-positive neurons in laminae I, IIo, and X and in half of NK1R-positive neurons in laminae III-V. SP-induced internalization was abolished by the specific NK1R antagonist L-703,606 (1 microM). Stimulating the dorsal root with long-duration (0.4 msec) pulses evoked EPSPs in dorsal horn neurons with latencies consistent with the conduction speed of A partial differential- and C-fibers. High-frequency (100 Hz) stimulation of the dorsal root with these pulses induced NK1R internalization in neurons in laminae I-IIo of the stimulated side of the slice but not in the contralateral side or in other laminae. Stimulation at lower frequencies (1 and 10 Hz) failed to elicit significant internalization, suggesting that the release of SP is frequency-dependent. Internalization produced by the 100 Hz tetanus was mimicked by NMDA and blocked by an NMDA antagonist, 2-amino-5-phosphonopentanoic acid, but not by the AMPA and kainate antagonist CNQX. The NK1R antagonist L-703,606 abolished the internalization produced by 100 Hz stimulation or NMDA. Therefore, the release of SP in the dorsal horn appears to be controlled by NMDA receptors.