Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 and, by unknown mechanisms, induce widespread inflammation. We found that a large proportion of primary spinal afferent neurons, which express proteinase-activated receptor 2, also contain the proinflammatory neuropeptides calcitonin gene-related peptide and substance P. Trypsin and tryptase directly signal to neurons to stimulate release of these neuropeptides, which mediate inflammatory edema induced by agonists of proteinase-activated receptor 2. This new mechanism of protease-induced neurogenic inflammation may contribute to the proinflammatory effects of mast cells in human disease. Thus, tryptase inhibitors and antagonists of proteinase-activated receptor 2 may be useful anti-inflammatory agents.

Although a role for the gastric and intestinal mucosa in molecular sensing has been known for decades, the initial molecular recognition events that sense the chemical composition of the luminal contents has remained elusive. Here we identified putative taste receptor gene transcripts in the gastrointestinal tract. Our results, using reverse transcriptase-PCR, demonstrate the presence of transcripts corresponding to multiple members of the T2R family of bitter taste receptors in the antral and fundic gastric mucosa as well as in the lining of the duodenum. In addition, cDNA clones of T2R receptors were detected in a rat gastric endocrine cell cDNA library, suggesting that these receptors are expressed, at least partly, in enteroendocrine cells. Accordingly, expression of multiple T2R receptors also was found in STC-1 cells, an enteroendocrine cell line. The expression of ␣ subunits of G proteins implicated in intracellular taste signal transduction, namely G␣gust, and G␣t-2, also was demonstrated in the gastrointestinal mucosa as well as in STC-1 cells, as revealed by reverse transcriptase-PCR and DNA sequencing, immunohistochemistry, and Western blotting. Furthermore, addition of compounds widely used in bitter taste signaling (e.g., denatonium, phenylthiocarbamide, 6-n-propil-2-thiouracil, and cycloheximide) to STC-1 cells promoted a rapid increase in intracellular Ca 2؉ concentration. These results demonstrate the expression of bitter taste receptors of the T2R family in the mouse and rat gastrointestinal tract.stomach ͉ intestine ͉ gustducin ͉ transducin T he gustatory system has been selected during evolution to detect nutritive and beneficial compounds as well as harmful or toxic substances (1, 2). In particular, bitter taste has evolved as a central warning signal against the ingestion of potentially toxic substances (3). Recently, a large family of bitter taste receptors (T2Rs) expressed in specialized neuroepithelial taste receptor cells organized within taste buds in the tongue has been identified in humans and rodents (4-6). These putative taste receptors, which belong to the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor superfamily characterized by seven putative transmembrane domains, are distantly related to V1R vomeronasal receptors and opsins (5). Genetic and biochemical evidence indicate that specific G␣ subunits, gustducin (G␣ gust ) and transducin (G␣ t ), mediate bitter and sweet gustatory signals in the taste buds of the lingual epithelium (7-11).Outside the tongue, expression of G␣ gust also has been localized to gastric (12) and pancreatic (13) cells, suggesting that a taste-sensing mechanism also may exist in the gastrointestinal (GI) tract. However, not all cells that express G␣ gust also coexpress members of the T2R family of receptors (5). For example, most G␣ gust -positive taste receptor cells in the lingual fungiform papillae are T2R-negative, implying that G␣ gust also could mediate signaling through other receptors (9). To establish that the gastric an...

A nerve process grows by inserting new membrane material at its advancing tip, the growth cone. In embryonic cell culture and in embryos of Xenopus laevis, many growth cones establish functional synaptic transmission within minutes after contact with muscle cells. The rapidity of synapse formation suggests that the growth cone may have already acquired the appropriate neurotransmitter and the machinery for transmitter release before encountering the target cell. Here, we have used a patch of outside-out embryonic muscle membrane formed with gigaohm seal at the tip of a micropipette as an extracellular probe for the presence of channel-activating substances near the growth cones of the isolated Xenopus embryonic neurones in culture. We report that single-channel activity resembling that of muscle acetylcholine receptor channels was induced when the probe was positioned near the growth cones of 50% of the neurones, suggesting the spontaneous release of acetylcholine (ACh) from these growth cones. The release of material from growth cones may occur as a consequence of the incorporation of new membrane during neurite extension; it may also have a role in the interaction between the growth cone and its immediate environment.

Protein kinase D (PKD)/protein kinase C (PKC) is a serine/threonine protein kinase that can be activated by physiological stimuli like growth factors, antigen-receptor engagement and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires PKC activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular translocation of a green fluorescent protein-tagged PKD was analyzed by real-time visualization in fibroblasts and epithelial cells stimulated with bombesin, a GPCR agonist. We found that bombesin induced a rapidly reversible plasma membrane translocation of green fluorescent protein-tagged PKD, an event that can be divided into two distinct mechanistic steps. The first step, which is exclusively mediated by the cysteine-rich domain in the N terminus of PKD, involved its translocation from the cytosol to the plasma membrane. The second step, i.e. the rapid reverse translocation of PKD from the plasma membrane to the cytosol, required its catalytic domain and surprisingly PKC activity. These findings provide evidence for a novel mechanism by which PKC coordinates the translocation and activation of PKD in response to bombesininduced GPCR activation.

1 Thrombin, generated in the circulation during injury, cleaves proteinase-activated receptor 1 (PAR1) to stimulate plasma extravasation and granulocyte in®ltration. However, the mechanism of thrombin-induced in¯ammation 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. 2 PAR1 was detected in small diameter neurons known to contain SP in rat dorsal root ganglia by immunohistochemistry and in situ hybridization. 3 Thrombin and the PAR1 agonist TFLLR-NH 2 (TF-NH 2 ) increased [Ca 2+ ] i 450% of cultured neurons (EC 50 s 24 mu ml 71 and 1.9 mM, respectively), assessed using Fura-2 AM. The PAR1 agonist completely desensitized responses to thrombin, indicating that thrombin stimulates neurons through PAR1. 4 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. 5 In wild-type but not PAR1 7/7 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. 6 Thus, thrombin cleaves PAR1 on primary spinal a erent 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.

(8,9). In addition to its role as sensor of [Ca 2ϩ ] o , the CaR is also stimulated by aromatic amino acids (10) that, like [Ca 2ϩ ] o , induce striking and lasting CaR-mediated [Ca 2ϩ ] i oscillations (9, 11). However, the patterns of [Ca 2ϩ ] i oscillations induced by these agonists are different. Aromatic amino acid stimulation of the CaR induces repetitive, low frequency [Ca 2ϩ ] i spikes that return to the base-line level, a pattern known as transient oscillations. In contrast, [Ca 2ϩ ] o -elicited CaR activation produces high frequency sinusoidal oscillations upon a raised plateau level of [Ca 2ϩ ] i (9, 11). The amplitude, frequency, and duration of [Ca 2ϩ ] i oscillations are increasingly recognized as encoding important information for a variety of biological processes, and, consequently, there is intense interest in understanding the underlying mechanisms (12).Our previous results produced several lines of evidence indicating that PKCs negatively regulate the frequency of [Ca 2ϩ ] i oscillations induced by activation of the CaR by increases in [Ca 2ϩ ] o (11). We hypothesized that periodic phosphorylation of the CaR by PKCs provides the negative feedback needed to cause [Ca 2ϩ ] o -induced sinusoidal [Ca 2ϩ ] i oscillations. Intriguingly, the transient [Ca 2ϩ ] i oscillations produced by the CaR in response to amino acid stimulation appear to be mediated by a different pathway, but the mechanism(s) involved remained poorly understood.In the present study, we examined whether sinusoidal and transient [Ca 2ϩ ] i oscillations produced by the CaR in response to Ca 2ϩ or L-phenylalanine are mediated by different pathways. Using real time imaging of changes in phosphatidylinositol 4,5-biphosphate hydrolysis and generation of Ins(1,4,5)P 3 in single cells, we found that [Ca 2ϩ ] o -induced CaR activation

The protein kinase D (PKD) family consists of three serine/threonine kinases: PKC/PKD, PKD2, and PKC/PKD3. Whereas PKD has been the focus of most studies, virtually nothing is known about the effect of G protein-coupled receptor agonists (GPCR) on the regulatory properties and intracellular distribution of PKD3. Consequently, we examined the mechanism that mediates its activation and intracellular distribution. GPCR agonists induced a rapid activation of PKD3 by a protein kinase C (PKC)-dependent pathway that leads to the phosphorylation of the activation loop of PKD3. Comparison of the steady-state distribution of endogenous or tagged PKD3 versus PKD and PKD2 in unstimulated cells indicated that whereas PKD and PKD2 are predominantly cytoplasmic, PKD3 is present both in the nucleus and cytoplasm. This distribution of PKD3 results from its continuous shuttling between both compartments by a mechanism that requires a nuclear import receptor and a competent CRM1-nuclear export pathway. Cell stimulation with the GPCR agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD3 that is PKCdependent. Interestingly, the nuclear accumulation of PKD3 can be dramatically enhanced in response to its activation. Thus, this study demonstrates that the intracellular distribution of PKD isoenzymes are distinct, and suggests that their signaling properties are regulated by differential localization.

Donor specific HLA antibodies significantly lower allograft survival, but as yet there are no satisfactory therapies for prevention of antibody-mediated rejection. Intracapillary macrophage infiltration is a hallmark of antibody-mediated rejection, and macrophages are important in both acute and chronic rejection. The purpose of this study was to investigate the Fc-independent effect of HLA I antibodies on endothelial cell activation, leading to monocyte recruitment. We used an in vitro model to assess monocyte binding to endothelial cells in response to HLA I antibodies. We confirmed our results in a mouse model of antibody-mediated rejection, in which B6.RAG1-/- recipients of BALB/c cardiac allografts were passively transferred with donor specific MHC I antibodies. Our findings demonstrate that HLA I antibodies rapidly increase intracellular calcium and endothelial presentation of P-selectin, which supports monocyte binding. In the experimental model, donor specific MHC I antibodies significantly increased macrophage accumulation in the allograft. Concurrent administration of rPSGL-1-Ig abolished antibody-induced monocyte infiltration in the allograft, but had little effect on antibody-induced endothelial injury. Our data suggest that antagonism of P-selectin may ameliorate accumulation of macrophages in the allograft during antibody-mediated rejection.