Activation Of Proteinase-activated Receptors Research Articles

Involvement of PAR2 in inflammatory mediator release from human blood eosinophils

Proteinase Activated Receptors (PARs) are the members of G-protein-coupled receptor family and can be cleaved by certain serine proteases to expose a tethered ligand domain, which binds and activates the receptors to initiate multiple signaling cascades. There is some evidence that certain proteases may regulate target cells by activating PARs. There are many studies, in which PARs play important roles in inflammation. One study indicated that PAR2 inhibition and deletion significantly suppressed the degree of inflammation due to decreased IL-6 and IL-1 levels. Another study also showed that PARs activation could mediate reactive oxygen species production and MAPK signaling leading to alveolar inflammation. In addition, platelet-derived CAPN1 can trigger the vascular inflammation associated with diabetes via cleavage of PAR1 and the release of TNF from the endothelial cell surface, and sarsasapogenin may alleviate diabetic nephropathy by the downregulation of PAR1. Another Phellodendron amurense bark extract can suppress the particulate matter-induced Ca2+ influx caused by direct action upon PAR2, alleviating inflammation and maintaining homeostatic levels of cell adhesion components. There are also other two antagonists of I-287 and GB88, which can reduce the PAR2-mediated inflammatory reaction. In this study, we tested expression of PARs and IL-5, IL-6, RANTES and ECP release from human blood eosinophils using different enzymes and PAR agonists. The expression of PARs was assessed in human blood eosinophils by flow cytometry and RT-PCR, and the levels of cytokine and eosinophil cationic protein (ECP) in the cultured supernatants were determined with ELISA kits. Flow cytometry shows that human eosinophils express PAR2 protein and do not express PAR1, PAR3 and PAR4 proteins. RT-PCR analysis revealed expression of PAR2 and PAR3 genes in human eosinophils. Tryptase, trypsin and elastase can induce significant IL-5, IL-6 and ECP release. Trypsin and elastase may also stimulate RANTES secretion, but tryptase cannot induce the RANTES secretion. Tryptase, trypsin and elastase-induced cytokine and ECP release from human blood eosinophils most likely occurs via activation of PAR2.

Molecular mechanisms regulating Proteinase‐Activated Receptors (PARs)

Proteinase-activated receptors (PARs) are a four-member family of G protein-coupled receptors defined by their irreversible proteolytic mechanism of activation. PARs have emerged as important regulators of various physiological responses and are implicated in numerous pathological conditions. Importantly, PAR1 and PAR4 are critical regulators of platelet function, while PAR2 is well established as a driver of inflammatory responses. PAR-targeted drug development efforts are therefore of great interest. In this review, we provide an overview of recent advances in our understanding of molecular mechanisms underlying PAR activation, effector interaction, and signaling. We also provide an overview of the diverse proteolytic enzymes that are now established as PAR regulators and describe the ability of different enzymes to elicit biased signaling through PARs. Finally, we highlight recent advances in the development of PAR-targeted pharmacological agents and discuss recent structure-activity relationship studies.

Proteinase-activated receptors (PARs) as targets for antiplatelet therapy.

Since the identification of the proteinase-activated receptor (PAR) family as mediators of serine protease activity in the 1990s, there has been tremendous progress in the elucidation of their pathophysiological roles. The development of drugs that target PARs has been the focus of many laboratories for the potential treatment of thrombosis, cancer and other inflammatory diseases. Understanding the mechanisms of PAR activation and G protein signalling pathways evoked in response to the growing list of endogenous proteases has yielded great insight into receptor regulation at the molecular level. This has led to the development of new selective modulators of PAR activity, particularly PAR1. The mixed success of targeting PARs has been best exemplified in the context of inhibiting PAR1 as a new antiplatelet therapy. The development of the competitive PAR1 antagonist, vorapaxar (Zontivity), has clearly shown the value in targeting PAR1 in acute coronary syndrome (ACS); however the severity of associated bleeding with this drug has limited its use in the clinic. Due to the efficacy of thrombin acting via PAR1, strategies to selectively inhibit specific PAR1-mediated G protein signalling pathways or to target the second thrombin platelet receptor, PAR4, are being devised. The rationale behind these alternative approaches is to bias downstream thrombin activity via PARs to allow for inhibition of pro-thrombotic pathways but maintain other pathways that may preserve haemostatic balance and improve bleeding profiles for widespread clinical use. This review summarizes the structural determinants that regulate PARs and the modulators of PAR activity developed to date.

Identification of a Prostate Cancer Cell Proteinase Activated Receptor/MMP Signaling Axis

HypothesisProteinase Activated Receptors (PARs) are G‐Protein‐Coupled Receptors activated by the proteolytic unmasking of a cryptic tethered ligand. We proposed that prostate cancer cells produce PAR‐regulating enzymes.MethodsWe monitored PC3, DU145 and LNCaP cell supernatant cleavage of PARs in intact cells by fluorescence imaging using N‐ and C‐ terminus dual‐fluorophore‐tagged PARs. Loss of the N‐terminal tag denotes receptor cleavage‐activation. We also used N‐terminal nano‐luciferase‐tagged PARs that, via luciferase release, document PAR‐cleaving proteinase activity in cell‐derived supernatants. MAPKinase and transwell migration assays were used to monitor PAR activation in PC3 cells.ResultsActivation of PARs 1 & 2 triggered MAPKinase signaling and cancer cell migration. Distinct PAR‐cleaving proteinases in conditioned media from all three prostate cancer cell lines were observed with the N‐Luc‐Luciferase assay, with differential inhibition by matrix metalloproteinase inhibitors and soya trypsin inhibitor. Further, when expressed in PC3 cells, the dual‐fluorophore‐tagged PAR1 was seen in a constitutively cleaved‐active state, with an absent N‐terminus.ConclusionOur data show that prostate cancer‐derived cells secrete PAR‐regulating proteinases that, via an autocrine process, can regulate PAR function in the tumour microenvironment. Since PAR activation triggers prostate cell migration and MAPKinase, PAR antagonists and inhibitors of PAR‐activating proteinases represent therapeutic targets for treating prostate cancer.FundingProstate Cancer Canada Movember Discovery grant.

The contribution of proteinase-activated receptors to intracellular signaling, transcellular transport and autophagy in Alzheimer's disease.

The etiopathogenesis of Alzheimer´s disease is characterized by beta amyloid Aβ(1-42) toxic fragment aggregation and its association with impaired autophagy. In mitochondria, chronic damage due to transport and enzymatic processes together with the production of reactive oxygen species (ROS) are followed by the subsequent accumulation of Aβ in the form of senile plaques and the accumulation of hyperphosphorylated tau protein in intracellular deposits called tangles. Proteinase-activated receptors (PARs), members of the G protein-coupled receptor (GPCR) family, facilitate and modulate the transcellular transport and distribution of a variety of subcellular molecular components to the lysosomal system and, thus, influence their degradation. A review of the data shows that the activation or inhibition of PARs leads to changes in the process of autophagy, which may influence ROS production and Aβ (1-42) degradation in lysosomes and result in AD pathogenesis.

Blockade of proteinase-activated receptor 4 inhibits neutrophil recruitment in experimental inflammation in mice.

The activation of proteinase-activated receptors (PARs) has been implicated in the development of important hallmarks of inflammation, including in vivo leukocyte recruitment; however, its role in the regulation of leukocyte migration in response to inflammatory stimuli has not been elucidated until now. Here, we examined the effects of the PAR4 antagonist YPGKF-NH 2 (tcY-NH2) on neutrophil recruitment in experimentally induced inflammation. BALB/c mice were intrapleurally injected with tcY-NH2 (40ng/kg) prior to intrapleural injection of carrageenan (Cg) or neutrophil chemoattractant CXCL8; the number of infiltrating neutrophils was evaluated after 4h, and KC production was assessed at different times after Cg injection. Neutrophil adhesion and rolling cells were studied using a brain circulation preparation 4h after the Cg or CXCL8 challenge in tcY-NH2-treated mice. PAR4 blockade inhibited CXCL8- and Cg-induced neutrophil migration into the pleural cavity of BALB/c mice and reduced neutrophil rolling and adherence. Surprisingly, PAR4 blockade increased the level of KC in response to carrageenan. These results demonstrated that PAR4 blockade impairs neutrophil migration in vivo, suggesting that PAR4 plays an important role in the regulation of inflammation, at least in part because of its ability to inhibit the actions of the neutrophil chemoattractant CXCL8.

Biased signalling and proteinase‐activated receptors (PARs): targeting inflammatory disease

Although it has been known since the 1960s that trypsin and chymotrypsin can mimic hormone action in tissues, it took until the 1990s to discover that serine proteinases can regulate cells by cleaving and activating a unique four-member family of GPCRs known as proteinase-activated receptors (PARs). PAR activation involves the proteolytic exposure of its N-terminal receptor sequence that folds back to function as a 'tethered' receptor-activating ligand (TL). A key N-terminal arginine in each of PARs 1 to 4 has been singled out as a target for cleavage by thrombin (PARs 1, 3 and 4), trypsin (PARs 2 and 4) or other proteases to unmask the TL that activates signalling via Gq , Gi or G12 /13 . Similarly, synthetic receptor-activating peptides, corresponding to the exposed 'TL sequences' (e.g. SFLLRN-, for PAR1 or SLIGRL- for PAR2) can, like proteinase activation, also drive signalling via Gq , Gi and G12 /13 , without requiring receptor cleavage. Recent data show, however, that distinct proteinase-revealed 'non-canonical' PAR tethered-ligand sequences and PAR-activating agonist and antagonist peptide analogues can induce 'biased' PAR signalling, for example, via G12 /13 -MAPKinase instead of Gq -calcium. This overview summarizes implications of this 'biased' signalling by PAR agonists and antagonists for the recognized roles the PARs play in inflammatory settings.

Targeting factor Xa and thrombin: impact on coagulation and beyond

Great advances have been made in recent years in understanding the haemostatic system and the molecular and cellular basis of thrombus formation. Although directly targeting factorXa or thrombin (factor IIa) for effective anticoagulation is now well established, evidence has emerged suggesting that factor Xa and thrombin are involved in other physiological and pathophysiological cellular processes, including inflammation. These non-haemostatic activities of factor Xa and thrombin are predominantly mediated via the activation of proteinase-activated receptors. Studies have indicated a potential role of coagulation proteins (including factor Xa and thrombin) in the progression of disease conditions such as atherothrombosis. Preclinical studies have provided evidence for the effects of direct factor Xa or direct thrombin inhibition beyond anticoagulation, including anti-inflammatory activities and atherosclerotic plaque stabilisation. In this article, the non-haemostatic activities of factor Xa and thrombin and the effects of direct inhibition of these coagulation factors on these activities are summarised. In addition, the potential roles of factor Xa and thrombin in atherosclerosis and atherothrombosis are explored and the cardiovascular profiles of rivaroxaban, apixaban and dabigatran etexilate observed in phaseIII clinical studies are discussed.

Cofactoring and Dimerization of Proteinase-Activated Receptors

Proteinase-activated receptors (PARs) are G protein-coupled receptors that transmit cellular responses to extracellular proteases and have important functions in vascular physiology, development, inflammation, and cancer progression. The established paradigm for PAR activation involves proteolytic cleavage of the extracellular N terminus, which reveals a new N terminus that functions as a tethered ligand by binding intramolecularly to the receptor to trigger transmembrane signaling. Most cells express more than one PAR, which can influence the mode of PAR activation and signaling. Clear examples include murine PAR3 cofactoring of PAR4 and transactivation of PAR2 by PAR1. Thrombin binds to and cleaves murine PAR3, which facilitates PAR4 cleavage and activation. This process is essential for thrombin signaling and platelet activation, since murine PAR3 cannot signal alone. Although PAR1 and PAR4 are both competent to signal, PAR1 is able to act as a cofactor for PAR4, facilitating more rapid cleavage and activation by thrombin. PAR1 can also facilitate PAR2 activation through a different mechanism. Cleavage of the PAR1 N terminus by thrombin generates a tethered ligand domain that can bind intermolecularly to PAR2 to activate signaling. Thus, PARs can regulate each other's activity by localizing thrombin when in complex with PAR3 and PAR4 or by cleaved PAR1, providing its tethered ligand domain for PAR2 activation. The ability of PARs to cofactor or transactivate other PARs would necessitate that the two receptors be in close proximity, likely in the form of a heterodimer. Here, we discuss the cofactoring and dimerization of PARs and the functional consequences on signaling.

Proteinase-Activated Receptor-4 Plays a Major Role in the Recruitment of Neutrophils Induced by Trypsin or Carrageenan during Pleurisy in Mice

Background/Aims: The activation of proteinase-activated receptors (PARs) has been implicated in the development of important hallmarks of inflammation, including in vivo leukocyte recruitment. Here, we examined the effects of aprotinin, a potent inhibitor of trypsin proteinase and the kallikrein-kinin system, and the PAR-4 antagonist YPGKF-NH₂ (tcY-NH₂) on neutrophil recruitment in response to carrageenan and trypsin in the pleural cavity of mice. Methods: BALB/c mice were intrapleurally injected with trypsin or PAR-4-activating peptide AY-NH₂, pretreated with aprotinin or tcY-NH₂ (1 µg/cavity) prior to an intrapleural injection of trypsin or carrageenan, or pretreated with leukotriene B₄ antagonist U-75302 (3 µg/cavity) prior to a trypsin injection. The number of infiltrating neutrophils was evaluated after 4 h. Results: PAR-4-activating peptide AY-NH₂ and trypsin-induced neutrophil recruitment was inhibited by aprotinin, tcY-NH₂ or U-75302. Aprotinin and tcY-NH₂ also inhibited neutrophil recruitment induced by carrageenan. Conclusion: These data suggest a key role for PAR-4 in mediating neutrophil recruitment in a mouse model of pleurisy induced by the activity of trypsin or trypsin-like enzymes.

Allergen‐derived proteinases: Isolation, characterization and signaling via proteinase‐activated receptors (PARs)

ObjectiveTo identify proteolytic enzymes in insect and mould asthma allergens and assess their ability to signal via the PARs.MethodsAllergen‐derived trypsin‐like enzymes known to contribute to lung inflammation were visualized (western blot) with serine proteinase activity‐based probes (ABP). Enzymes were purified using ion‐exchange chromatography and analyzed for their ABP reactivity & substrate‐inhibitor kinetic profiles. Each enzyme was tested for its activation of PAR–dependent calcium signaling in PAR1 & PAR2‐expressing KNRK cells. ABP‐labeled enzymes were isolated by avidin affinity chromatography and their peptide sequences determined (mass spectrometry).ResultsBoth allergens contained ABP‐labeled proteinases with MWs of 20–26 kDa. Each enzyme had distinct kinetic substrate (Km) and inhibitor (Ki) profiles. The enzymes all caused PAR2 calcium signaling, and differentially activated PAR1. Sequencing revealed that the allergen proteinases were homologous with previously described trypsin‐like enzymes.ConclusionsDiverse allergens contain multiple biochemically distinct trypsin‐like proteinases that are able to target PARs 1 and 2. Thus, PARs and their activating allergen proteinases may be attractive therapeutic targets for the treatment of allergen‐induced asthma.Support: Canadian Institutes of Health Research, NIH USA and Lung Association of Alberta & NWT

Kallikrein-related peptidases: proteolysis and signaling in cancer, the new frontier

The exact mechanism(s) by which kallikrein-related peptidases (KLKs) function, their levels of activity and their potential endogenous targets in vivo have only recently begun to be revealed. Our group and others have shown that KLKs can have hormonal properties by signaling via proteinase-activated receptors (PARs), a family of G-protein-coupled receptors. Signals by PAR(1), PAR(2), and PAR(4) can regulate calcium release or mitogen-activated protein kinase activation and lead to platelet aggregation, vascular relaxation, cell proliferation, cytokine release, and inflammation. We have further documented the presence of active KLK6 and 10 (by activity-based ELISA or proteomics) and the presence of proteinase inhibitors, such as alpha(1)-antitrypsin, in cancer-derived fluids. We suggest that tumors and inflamed tissues can release active KLKs, which are under tight regulation by proteinase inhibitors. These enzymes can potentially control cell/tissue behavior by regulating PAR activation in specific settings and disease stages.

Proteinase‐activated receptors induce interleukin‐8 expression by intestinal epithelial cells through ERK/RSK90 activation and histone acetylation

Proteinase-activated receptors (PARs) are involved in both inflammation and tumorigenesis in epithelial cells. Interleukin (IL)-8 is a potent chemoattractant and is also involved in angiogenesis. The molecular mechanism whereby PARs induce epithelial IL-8 expression is not known. In HT-29 colonic epithelial cells, PAR(1) or PAR(2) agonists stimulated the expression of IL-8 through a NF-kappaB-dependent pathway without inducing IkappaB degradation and disassociation of IkappaB from NF-kappaB. Further studies revealed that PAR activation induced the phosphorylation of p65 at Ser-276 in the nucleus, which increased the recruitment of histone acetyltransferase (HAT) p300 to p50. Inhibition of ERK activation completely blocked PAR-induced IL-8 expression, phosphorylation of p65 and HAT activity. We also demonstrated that RSK p90 was the downstream kinase that mediated ERK-induced nuclear p65 phosphorylation. In conclusion, activation of either PAR(1) or PAR(2) stimulated the transcriptional up-regulation of IL-8 in HT-29 colonic epithelial cells through a pathway that involved ERK/RSK p90, NF-kappaB phosphorylation, and HAT activity. These studies provide evidence of a new role for serine proteinases and PARs in the regulation of gene expression in colonic inflammation and tumorigenesis.

Coagulation factor Xa signaling: the link between coagulation and inflammatory bowel disease?

Inflammatory bowel disease (IBD) is characterized by activation of the coagulation cascade and it has long been suspected that coagulation is an essential component of this still largely idiopathic group of diseases. The realization that coagulation factors are not only passive mediators in the propagation of coagulation, but also actively engage different cell types by activating proteinase-activated receptors (PARs) has provided mechanistic insight into how coagulation cascade propagation might participate in the progression of IBD. The emergence of PAR(2) as a key player in the progression of IBD has focused attention on its agonist, coagulation factor Xa (FXa). Recent findings on FXa and PAR(2) link the coagulation cascade to the progression of IBD. Here, we propose that FXa-induced PAR(2) activation has an important role in orchestrating intestinal inflammatory and fibroproliferative responses, and that PAR(2) might provide a novel therapeutic target for the management of IBD.

Prostatic trypsin-like kallikrein-related peptidases (KLKs) and other prostate-expressed tryptic proteinases as regulators of signalling via proteinase-activated receptors (PARs)

The prostate is a site of high expression of serine proteinases including members of the kallikrein-related peptidase (KLK) family, as well as other secreted and membrane-anchored serine proteinases. It has been known for some time that members of this enzyme family elicit cellular responses by acting directly on cells. More recently, it has been recognised that for serine proteinases with specificity for cleavage after arginine and lysine residues (trypsin-like or tryptic enzymes) these cellular responses are often mediated by cleavage of members of the proteinase-activated receptor (PAR) family--a four member sub-family of G protein-coupled receptors. Here, we review the expression of PARs in prostate, the ability of prostatic trypsin-like KLKs and other prostate-expressed tryptic enzymes to cleave PARs, as well as the prostate cancer-associated consequences of PAR activation. In addition, we explore the dysregulation of trypsin-like serine proteinase activity through the loss of normal inhibitory mechanisms and potential interactions between these dysregulated enzymes leading to aberrant PAR activation, intracellular signalling and cancer-promoting cellular changes.

Kallikreins and proteinase-mediated signaling: proteinase-activated receptors (PARs) and the pathophysiology of inflammatory diseases and cancer

Proteinases such as thrombin and trypsin can affect tissues by activating a novel family of G protein-coupled proteinase-activated receptors (PARs 1-4) by exposing a 'tethered' receptor-triggering ligand (TL). Work with synthetic TL-derived PAR peptide sequences (PAR-APs) that stimulate PARs 1, 2 and 4 has shown that PAR activation can play a role in many tissues, including the gastrointestinal tract, kidney, muscle, nerve, lung and the central and peripheral nervous systems, and can promote tumor growth and invasion. PARs may play roles in many settings, including cancer, arthritis, asthma, inflammatory bowel disease, neurodegeneration and cardiovascular disease, as well as in pathogen-induced inflammation. In addition to activating or disarming PARs, proteinases can also cause hormone-like effects via PAR-independent mechanisms, such as activation of the insulin receptor. In addition to proteinases of the coagulation cascade, recent data suggest that members of the family of kallikrein-related peptidases (KLKs) represent endogenous PAR regulators. In summary: (1) proteinases are like hormones, signaling in a paracrine and endocrine manner via PARs or other mechanisms; (2) KLKs must now be seen as potential hormone-like PAR regulators in vivo; and (3) PAR-regulating proteinases, their target PARs, and their associated signaling pathways appear to be novel therapeutic targets.

Procoagulant signalling mechanisms in lung inflammation and fibrosis: novel opportunities for pharmacological intervention?

There is compelling evidence that uncontrolled activation of the coagulation cascade following lung injury contributes to the development of lung inflammation and fibrosis in acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and fibrotic lung disease. This article reviews our current understanding of the mechanisms leading to the activation of the coagulation cascade in response to lung injury and the evidence that excessive procoagulant activity is of pathophysiological significance in these disease settings. Current evidence suggests that the tissue factor-dependent extrinsic pathway is the predominant mechanism by which the coagulation cascade is locally activated in the lungs of patients with ALI/ARDS and pulmonary fibrosis. Whilst, fibrin deposition might contribute to the pathophysiology of ALI/ARDS following systemic insult; current evidence suggests that the cellular effects mediated via activation of proteinase-activated receptors (PARs) may be of particular importance in influencing inflammatory and fibroproliferative responses in experimental models involving direct injury to the lung. In this regard, studies in PAR(1) knockout mice have shown that this receptor plays a major role in orchestrating the interplay between coagulation, inflammation and lung fibrosis. This review will focus on our current understanding of excessive procoagulant signalling in acute and chronic lung injury and will highlight the novel opportunities that this may present for therapeutic intervention.

Proteinases, proteinase-activated receptors (PARs) and the pathophysiology of cancer and diseases of the cardiovascular, musculoskeletal, nervous and gastrointestinal systems

Proteinases like thrombin, trypsin and tissue kallikreins are now known to regulate cell signaling by cleaving and activating a novel family of G-protein-coupled proteinase-activated receptors (PARs 1 to 4) via exposure of a 'tethered' receptor-triggering ligand. On their own, short synthetic peptides based on the 'tethered ligand' sequences of the PARs (PAR-APs) can, in the absence of receptor proteolysis, selectively activate PARs 1, 2 and 4 and cause physiological responses both in vitro and in vivo. Using the PAR-APs as probes in vivo, it has been found that PAR activation can affect the vascular, renal, respiratory, gastrointestinal, musculoskeletal and nervous systems (both central and peripheral) and can promote cancer metastasis and invasion. The responses triggered by PARs 1, 2 and 4 are in keeping with an innate immune inflammatory response, ranging from vasodilatation to intestinal inflammation, increased cytokine production and increased nociception. Thus, PARs have been implicated in a number of disease states including cancer and inflammation of the cardiovascular, respiratory, musculoskeletal, gastrointestinal and nervous systems. Furthermore, PAR-regulating proteinases have been implicated in pathogen-induced inflammation. The identities of the proteinases that regulate PARs in these pathological settings in vivo have yet to be explored in depth. In addition to activating or dis-arming PARs, proteinases can also cause hormone-like effects by signaling mechanisms that do not involve the PARs and that may be as important as the activation of PARs. Thus, the working hypotheses of this article are: (1) that proteinases in general must now be considered as 'hormone-like' messengers that can signal either via PARs or other mechanisms and (2) that the PARs themselves, their activating serine proteinases and their associated signaling pathways can be considered as attractive targets for therapeutic drug development.

Kallikrein-mediated activation of PARs in inflammation and nociception

Kallikreins (KLKs) are secreted serine proteinases identified in a variety of cancers and in settings of inflammation.We have shown that KLKs in vitro can activate proteinase-activated receptors (PARs), a family of G-protein coupled receptors associated with inflammation. KLK14 activated both of PARs 2 and 4 and prevented thrombin from activating PAR1. On the other hand, KLK6 was a specific activator of PAR2. In an in vivo mouse inflammation model, kallikrein administration caused an oedema response comparable in magnitude and timing to that generated by trypsin. The oedema was accompanied by a decreased threshold of mechanical and thermal nociception. Our data demonstrate that by activating PARs 2 and 4 and by inactivating PAR1, widely-expressed kallikreins, like KLKs 6 and 14, may play a role in regulating the inflammatory response and pain perception.

Proteinase-activated receptors 1 and 2 in rat olfactory system: Layer-specific regulation of multiple signaling pathways in the main olfactory bulb and induction of neurite retraction in olfactory sensory neurons

Proteinase-activated receptors (PARs) are a family of four G protein–coupled receptors that are widely distributed in the CNS and involved in neural cell proliferation, differentiation and survival. The olfactory system undergoes continuous neurogenesis throughout life and may represent a critical target of PAR cellular actions. In the present study we investigated the functional activity of PAR 1 and PAR 2 in microdissected tissue preparations of olfactory nerve–glomerular layer (ON–GL), external plexiform layer (EPL) and granule cell layer (GRL) of the rat main olfactory bulb and in primary cultures of olfactory neuroepithelial cells. Activation of either PAR 1 or PAR 2 regulated multiple signaling pathways, including activation of pertussis-toxin sensitive G i/o proteins, inhibition of cyclic AMP formation, stimulation of G q/11-mediated phosphoinositide (PI) hydrolysis, phosphorylation of Ca 2+/calmodulin-dependent protein kinase II and activation of the monomeric G protein Rho, predominantly in ON–GL, whereas only activation of Rho was detected in the deeper layers. Olfactory nerve lesion by nasal irrigation with ZnSO 4 induced a marked decrease of PAR signaling in ON–GL. In primary cultures of olfactory neurons, double immunofluorescence analysis showed the localization of PAR 1 and PAR 2 in cells positive for olfactory-marker protein and neuron-specific enolase. Cell exposure to either nanomolar concentrations of thrombin and trypsin or PAR-activating peptides caused rapid neurite retraction. This study provides the first characterization of the laminar distribution of PAR 1 and PAR 2 signaling in rat olfactory bulb, demonstrates the presence of the receptors in olfactory sensory neurons and suggests a role of PARs in olfactory sensory neuron neuritogenesis.