Thrombin affects eosinophil migration via protease-activated receptor-1

BACKGROUND: Protease-activated receptors (PARs) are a unique class of G-protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. Although expression of the PAR1, which is typically activated by thrombin, on human eosinophils has been demonstrated, no effect of thrombin on eosinophil function has been shown yet. Thus we investigated whether thrombin affects eosinophil migration in vitro. METHODS: Eosinophils were obtained from venous blood of healthy donors. Cell migration was studied by micropore filter assays. Involvement of PARs in thrombin-dependent migration was tested functionally using selective agonist peptides for PARs and a cleavage blocking PAR1 antibody. RESULTS: Thrombin significantly stimulated eosinophil chemotaxis in a dose-dependent manner. This effect was mimicked by the PAR1 but not the PAR2 agonist and was reversed by the cleavage blocking PAR1 antibody. Checkerboard experiments indicated that eosinophil migration depends on the presence of thrombin in a concentration gradient. CONCLUSIONS: Data suggest that activation of PAR1 by thrombin stimulates directed migration of human eosinophils and thereby may affect eosinophils in tissue and allergic inflammation.     

Proteinase-activated receptor agonists stimulate the increase in intracellular Ca<Superscript>2+</Superscript> in cardiomyocytes and proliferation of cardiac fibroblasts from chick embryos

We studied activation of cultured cardiomyocytes and cardiac fibroblasts from chick embryos induced by agonists of PAR1 (thrombin and PAR1 peptide agonist) and PAR2 (trypsin, factor Xa, and peptide SLIGRL) by analyzing changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and cardiac fibroblast proliferation. Exposure of cardiomyocytes with thrombin induced immediate permanent dose-dependent increase in [Ca²⁺]_i. Ca²⁺ response decreased in a calcium-free medium. Peptide agonists of PAR1 and PAR2 also stimulated the increase in [Ca²⁺]_i in cardiomyocytes. Thrombin induced a short-term increase in [Ca²⁺]_i in cardiac fibroblasts and potentiated cell proliferation. PAR2 agonists trypsin and peptide SLIGRL stimulated proliferation of cardiac fibroblasts. Our results indicate that cardiomyocytes and cardiac fibroblasts from chick embryos have at least two types of PAR (types 1 and 2). __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 12, pp. 609–612, December, 2007     

Thrombin receptors and protease-activated receptor-2 in human placentation: receptor activation mediates extravillous trophoblast invasion in vitro

Proteolysis of the thrombin receptor, protease activated receptor-1 (PAR1), may enhance normal and pathological cellular invasion, and indirect evidence suggests that activation of PAR1 expressed by invasive extravillous trophoblasts (EVTs) influences human placentation. Here we describe PAR1, PAR2, and PAR3 protein distribution in the developing human placenta and implicate PAR1 and PAR2 activation in functions central to EVT invasion. PAR1, PAR2, and PAR3 are expressed in cultured 8- to 13-week-old EVTs, and in situ in 18- to 20-week-old placental syncytiotrophoblasts and invasive trophoblasts. Thrombin, but not the PAR2 agonist peptide SLIGKV, inhibited proliferation in cultured EVTs, although both agonists stimulated phosphoinositide hydrolysis and EVT invasion through Matrigel barriers. Thrombin-induced phosphoinositide hydrolysis was completely inhibited and the thrombin effect on proliferation was prevented when PAR1 cleavage was first blocked with specific monoclonal antibodies, indicating that PAR1 is the predominant thrombin receptor on EVTs. Together these results support a role for PAR1, and potentially PAR2 and PAR3 in the invasive phase of human placentation.     

The role of protease-activated receptors on the intracellular calcium ion dynamics of vascular smooth muscles, with special reference to cerebral arterioles

Protease-activated receptors (PARs) mediate cellular responses to various proteases in numerous cell types, including smooth muscles and the endothelium of blood vessels. To clarify whether the stimulation of PARs induces responses in smooth muscle cells of cerebral arterioles, intracellular Ca2+([Ca2+]i) dynamics and nitric oxide (NO) production during PARs stimulation were investigated in the rat cerebral arterioles by real-time confocal microscopy, since [Ca2+]i and NO are both key factors in the maintenance of strain in blood vessels. Testicular arterioles were also investigated for comparison. In smooth muscle cells of small cerebral arterioles (< 50 microm in diameter), thrombin and PAR1-activating peptide (AP) induced an increase in [Ca2+]i and contraction. The response to PAR1 activation was caused by Ca2+ mobilization from intracellular Ca2+ stores. Trypsin and PAR2-AP induced a decrease in [Ca2+]i in the cells which was considered to be mediated by endothelium-derived NO and/or by promoting a Ca2+ sequestration mechanism. PAR3- and 4-AP had little effect. In contrast to small cerebral arterioles, [Ca2+]i dynamics in smooth muscle cells of large cerebral arterioles (< 150 microm in diameter) or testicular arterioles remained unchanged during PARs activation. The effects of PARs activation on the [Ca2+]i dynamics and the contraction/relaxation of cerebral arterioles are also discussed in relation to the role of proteases in the regional tissue circulation of the brain.     

Neurotensin regulates intracellular calcium in ventral tegmental area astrocytes: evidence for the involvement of multiple receptors

Recent evidence suggests that some types of neurotensin receptors may be expressed by astrocytes. In order to explore the function of neurotensin receptors in astrocytes, the effect of a neurotensin receptor agonist, neurotensin(8-13), on intracellular Ca(2+) dynamics in mixed neuronal/glial cultures prepared from rat ventral tegmental area was examined. It was found that neurotensin(8-13) induces a long-lasting rise in intracellular Ca(2+) concentration in a subset of glial fibrilary acidic protein-positive glial cells. This response displays extensive desensitization and appears to implicate both intracellular and extracellular Ca(2+) sources. In the absence of extracellular Ca(2+), neurotensin(8-13) evokes only a short-lasting rise in intracellular Ca(2+). The neurotensin-evoked intracellular Ca(2+) accumulation is blocked by the phospholipase C inhibitor U73122 and by thapsigargin, suggesting that it is initiated by release of Ca(2+) from an inositol triphosphate-dependent store. The Ca(2+)-mobilizing action of neurotensin(8-13) in astrocytes is dependent on at least two receptors, because the response is blocked in part only by SR48692, a type 1 neurotensin receptor antagonist, and is blocked completely by SR142948A, a novel neurotensin receptor antagonist. The finding that the type 2 neurotensin receptor agonist levocabastine fails to mimic or alter the effects of neurotensin(8-13) on intracellular Ca(2+) makes it unlikely that the type 2 neurotensin receptor is involved.In summary, these results show that functional neurotensin receptors are present in cultured ventral tegmental area astrocytes and that their activation induces a highly desensitizing rise in intracellular Ca(2+). The pharmacological profile of this response suggests that a type 1 neurotensin receptor is involved but that another, possibly novel, non-type 2 neurotensin receptor is also implicated. If present in vivo, such signalling could be involved in some of the physiological actions of neurotensin.     

蛋白酶激活受体在炎性肠病大鼠模型迷走神经背侧运动核的表达及作用

目的： 研究炎性肠病大鼠模型的迷走神经背侧运动核（DMNV）蛋白酶激活受体（PAR-1,PAR-2）存在的情况,并阐明该受体激活的机制。方法： 制备20只炎性肠病的大鼠模型中取DMNV组织检测 PAR-1 和PAR-2;培养新出生的大鼠DMNV原代细胞,利用钙离子荧光探针Fura-2-AM检测PAR-1 和PAR-2及各种影响因素对细胞钙内流的影响。结果:凝血酶和其类似物PARP-1可以分别激活PAR-1出现最大钙离子内流223.3%±23.5%和145.6%±17.2%;胰蛋白酶和其类似物PARP-2 分别激活PAR-2出现最大钙离子内流242.7%±28.7%和236.7%±19.8%。使用1 μmol/L磷脂酶C抑制剂 U73312可以降低PAR-1激活的细胞钙离子内流140.1%±16.5%到20.7%±2.5%;降低PAR-2激活的钙离子内流225.4%±20.5%到45.4%±5.6%。钙离子抑制剂2APB可以降低PAR-1和PAR-2激活钙离子内流149.7%±13.4%和195.1%±21.5%分别到63.2%±4.3%和75.3%±13.5%。结论: 在DMNV中存在PAR-1和PAR-2,它们激活后通过磷脂酶C激活和1,4,5-三磷酸肌醇信号通路参与进行调解钙离子内流。     

Dopamine D1/5 receptor-mediated long-term potentiation of intrinsic excitability in rat prefrontal cortical neurons: Ca2+-dependent intracellular signaling

Prefrontal cortex (PFC) dopamine D1/5 receptors modulate long- and short-term neuronal plasticity that may contribute to cognitive functions. Synergistic to synaptic strength modulation, direct postsynaptic D1/5 receptor activation also modulates voltage-dependent ionic currents that regulate spike firing, thus altering the neuronal input-output relationships in a process called long-term potentiation of intrinsic excitability (LTP-IE). Here, the intracellular signals that mediate this D1/5 receptor-dependent LTP-IE were determined using whole cell current-clamp recordings in layer V/VI rat pyramidal neurons from PFC slices. After blockade of all major amino acid receptors (V(hold) = -65 mV) brief tetanic stimulation (20 Hz) of local afferents or application of the D1 agonist SKF81297 (0.2-50 microM) induced LTP-IE, as shown by a prolonged (>40 min) increase in depolarizing pulse-evoked spike firing. Pretreatment with the D1/5 antagonist SCH23390 (1 microM) blocked both the tetani- and D1/5 agonist-induced LTP-IE, suggesting a D1/5 receptor-mediated mechanism. The SKF81297-induced LTP-IE was significantly attenuated by Cd(2+), [Ca(2+)](i) chelation, by inhibition of phospholipase C, protein kinase-C, and Ca(2+)/calmodulin kinase-II, but not by inhibition of adenylate cyclase, protein kinase-A, MAP kinase, or L-type Ca(2+) channels. Thus this form of D1/5 receptor-mediated LTP-IE relied on Ca(2+) influx via non-L-type Ca(2+) channels, activation of PLC, intracellular Ca(2+) elevation, activation of Ca(2+)-dependent CaMKII, and PKC to mediate modulation of voltage-dependent ion channel(s). This D1/5 receptor-mediated modulation by PKC coexists with the previously described PKA-dependent modulation of K(+) and Ca(2+) currents to dynamically regulate overall excitability of PFC neurons.     

Contractility and Ca2+ signaling of smooth muscle cells in different generations of mouse airways

The control and mechanisms of airway smooth muscle cell (SMC) contraction were investigated with a sequential series of lung slices from different generations of the same airway from the cardiac lobe of the mouse lung. Airway contraction was measured by monitoring the changes in airway lumen area with phase-contrast microscopy. Changes in intracellular calcium concentration of the SMCs were studied with a custom-built confocal or two-photon microscope. The distribution of the airway SMCs and the muscarinic M(3) or 5-HT(2A) receptors was determined with immunofluorescence. Methacholine and 5-HT induced a concentration-dependent airway contraction and Ca(2+) oscillations within the SMCs of each airway generation. The airway contraction in response to the same agonist concentration was greater in the middle generation compared with the distal or proximal generations of the same airway. Similarly, the Ca(2+) oscillations varied in different generations of the same airway, with a slower frequency in the SMCs of the distal zone as compared with the middle or proximal zones of airways. By contrast, high KCl induced minimal contraction and very slow Ca(2+) oscillations throughout the whole intrapulmonary airway. The slower agonist-induced Ca(2+) oscillations in the distal zone correlated with a reduced expression of agonist receptors. The layer of SMCs increased in thickness in the middle and proximal zones. These results indicate that the contractility of airway SMCs varies at different positions along the same airway and that this response partially results from different Ca(2+) signaling and the total amount of the SMCs.     

Increase in intracellular Ca(2+) and calcitonin gene-related peptide release through metabotropic P2Y receptors in rat dorsal root ganglion neurons

We examined the effects of the activation of metabotropic P2Y receptors on the intracellular Ca(2+) concentration and the release of neuropeptide calcitonin gene-related peptide (CGRP) in isolated adult rat dorsal root ganglion neurons. In small-sized dorsal root ganglion neurons (soma diameter<30 microm) loaded with fura-2, a bath application of ATP (100 microM) evoked an increase in intracellular Ca(2+) concentration, while the removal of extracellular Ca(2+) partly depressed the response to ATP, thus suggesting that the ATP-induced increase in intracellular Ca(2+) concentration is due to both the release of Ca(2+) from intracellular stores and the influx of extracellular Ca(2+). Bath application of uridine 5'-triphosphate (UTP; 100 microM) also caused an increase in intracellular Ca(2+) concentration in small-sized dorsal root ganglion neurons and the P2 receptor antagonists suramin (100 microM) and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 10 microM) virtually abolished the response, indicating that the intracellular Ca(2+) elevation in response to UTP is mediated through metabotropic P2Y receptors. This intracellular Ca(2+) increase was abolished by pretreating the neurons with thapsigargin (100 nM), suggesting that the UTP-induced increase in intracellular Ca(2+) is primarily due to the release of Ca(2+) from endoplasmic reticulum Ca(2+) stores. An enzyme-linked immunosorbent assay showed that an application of UTP (100 microM) significantly stimulated the release of CGRP and that suramin (100 microM) totally abolished the response, suggesting that P2Y receptor-mediated increase in intracellular Ca(2+) is accompanied by CGRP release from dorsal root ganglion neurons.These results suggest that metabotropic P2Y receptors contribute to extracellular ATP-induced increase in intracellular Ca(2+) concentration and subsequent release of neuropeptide CGRP in rat dorsal root ganglion neurons.     

Synaptically activated calcium responses in dendrites of hippocampal oriens-alveus interneurons

Activation of metabotropic glutamate receptors (mGluRs) by agonists increases intracellular calcium levels ([Ca(2+)](i)) in interneurons of stratum oriens/alveus (OA) of the hippocampus. We examined the mechanisms that contribute to dendritic Ca(2+) increases in these interneurons during agonist activation of mGluRs and during synaptically evoked burst discharges, using simultaneous whole cell recordings and confocal Ca(2+) imaging in rat hippocampal slices. First, we found that the group I/II mGluR agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 100 microM) increased dendritic [Ca(2+)](i) and depolarized OA interneurons. Dendritic Ca(2+) responses were correlated with membrane depolarizations, but Ca(2+) responses induced by ACPD were larger in amplitude than those elicited by equivalent somatic depolarization. Next, we used linescans to measure changes in dendritic [Ca(2+)](i) during synaptically evoked burst discharges and somatically elicited repetitive firing in disinhibited slices. Dendritic Ca(2+) signals and electrophysiological responses were stable over repeated trials. Peak Ca(2+) responses were linearly related to number and frequency of action potentials in burst discharges for both synaptic and somatic stimulation, but the slope of the relationship was steeper for responses evoked somatically. Synaptically evoked [Ca(2+)](i) rises and excitatory postsynaptic potentials were abolished by antagonists of ionotropic glutamate receptors. The group I/II mGluR antagonist S-alpha-methyl-4-carboxyphenylglycine (500 microM) produced a significant partial reduction of synaptically evoked dendritic Ca(2+) responses. The mGluR antagonist did not affect synaptically evoked burst discharges and did not reduce either Ca(2+) responses or burst discharges evoked somatically. Therefore ionotropic glutamate receptors appear necessary for synaptically evoked dendritic Ca(2+) responses, and group I/II mGluRs may contribute partially to these responses. Dendritic [Ca(2+)](i) rises mediated by both ionotropic and metabotropic glutamate receptors may be important for synaptic plasticity and the selective vulnerability to excitotoxicity of OA interneurons.     

Ca2+ flux and signaling implications by nicotinic acetylcholine receptors in rat medial habenula

The fraction of inward current carried by Ca(2+) (FCa(2+)) through nicotinic acetylcholine receptors (nAChRs) on acutely isolated rat medial habenula (MHb) neurons was calculated from experiments that simultaneously monitored agonist-induced membrane currents and intracellular [Ca(2+)], measured with patch-clamp and indo-1 fluorescence, respectively. In physiological concentrations of extracellular Ca(2+) (2 mM) at -50 mV, the percentage of current carried by Ca(2+) was determined to be roughly 3-4%, which is in close agreement with measurements from other heteromeric nicotinic receptors expressed in peripheral tissue. Among factors that may have affected this measurement, such as Ca(2+) influx through voltage-gated Ca(2+) channels, the concentration of intracellular Ca(2+) buffer, and Ca(2+) sequestration and release from intracellular stores, only Ca(2+) uptake by mitochondria was shown to confound the analysis. Furthermore, we find that because of the high density of nAChRs on MHb cells, low concentrations of ACh (10 microM) and its hydrolysis product, choline (1 mM), can significantly elevate intracellular Ca(2+). Moreover, during persistent activation of nAChRs, the level of intracellular Ca(2+) is proportional to its extracellular concentration in the physiological range. Together, these findings support the suggestion that nAChRs may be capable of sensing low concentrations of diffusely released neurotransmitter and, in addition, transfer information about ongoing local synaptic activity by changes in extracellular Ca(2+).     

Development of testicular inflammation in the rat involves activation of proteinase-activated receptor-2

Mast cells are involved in early events crucial to inflammation and autoimmune disease. Recently, proteinase-activated receptor-2 (PAR(2)), a G-protein coupled receptor important to injury responses, was shown to be activated by mast cell tryptase. To investigate whether mast cells and PAR(2) are involved in the development and/or aggravation of testicular inflammation, we studied acute and chronic inflammatory models in the rat. In normal testes, PAR(2) was detected immunohistochemically in macrophages, in peritubular cells (PTCs) and in spermatid acrosomes. In experimentally induced autoimmune orchitis (EAO), PAR(2) was strongly upregulated in macrophages and peritubular-like cells, forming concentric layers around granulomas. Mast cells increased 10-fold in number, were more widely distributed throughout the interstitial tissue, and were partially degranulated. Isolated PTCs expressed functional PAR(2), responded to PAR(2) activation by phosphorylating extracellular signal-regulated kinases 1/2 (ERK1/2) and activating protein kinase c, and increased intracellular Ca(2+) concentrations as well as monocyte chemoattractant protein-1 (MCP-1), transforming growth factor beta(2) (TGFbeta(2)), and cyclooxygenase-2 (COX-2) mRNA expression. Expression of these inflammatory mediators, together with iNOS, also increased significantly in testes 50 days after EAO. In vivo, expression of cytokines and inflammatory mediators was upregulated after injection of recombinant tryptase (MCP-1, TGFbeta(2), and COX-2) and a specific PAR(2) peptide agonist (MCP-1, TGFbeta(2)) in the testis after 5 h. These results suggest that PAR(2) activation elicited on PTCs by mast cell tryptase contributes to acute testicular inflammation and that this pathogenetic mechanism may also play a role in autoimmune orchitis.     

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 PAR1 and PAR2 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 PAR1 or PAR2 regulated multiple signaling pathways, including activation of pertussis-toxin sensitive Gi/o proteins, inhibition of cyclic AMP formation, stimulation of Gq/11-mediated phosphoinositide (PI) hydrolysis, phosphorylation of Ca2+/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 ZnSO4 induced a marked decrease of PAR signaling in ON-GL. In primary cultures of olfactory neurons, double immunofluorescence analysis showed the localization of PAR1 and PAR2 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 PAR1 and PAR2 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.     

p-Nonylphenol induces endoplasmic reticulum stress-mediated apoptosis in neuronally differentiated PC12 cells

Endocrine disrupting chemicals (EDCs) induce estrogenic phenotypes in sexual organs and cells by chronic stimulation through binding to estrogen receptors. Although cell death may be induced instead of phenotypic change by EDCs in germ cells, the mechanism of the effect of EDCs in neuronal cells is still obscure. Here we report that p-nonylphenol, one of the EDCs, induced apoptosis with up-regulation of glucose-regulated protein 78 (GRP78) expression and activation of caspase-12, which are involved in endoplasmic reticulum (ER) stress specific phenomena, in NGF-treated neuronally differentiated PC12 cells. Moreover, we observed that p-nonylphenol increased the intracellular Ca(2+) concentration and p-nonylphenol-induced apoptosis was prevented when BAPTA-AM, a membrane-permeable Ca(2+) chelator, was added. Intriguingly, we also discovered that decreased phosphorylation of ERK1/2 was induced by p-nonylphenol in the presence of NGF, whereas p-nonylphenol alone did not induce phosphorylation of ERK1/2. These lines of evidence suggest that p-nonylphenol can induce ER stress-mediated apoptosis via increased intracellular Ca(2+) concentration, and can reduce ERK1/2 phosphorylation to attenuate the cell survival effect of NGF, in neuronally differentiated PC12 cells.     

Multiple P2 receptors contribute to a transient increase in intracellular Ca2+ concentration in ATP-stimulated rat brown adipocytes

Extracellular ATP in micromolar concentrations evokes a transient elevation in intracellular free Ca(2+) concentration ([Ca(2+)](i)), which arises primarily from a release of Ca(2+) from intracellular stores in rat brown adipocytes. We investigated the mechanisms underlying this transient nature of [Ca(2+)](i) elevation during exposure to ATP by using fura-2 fluorescence measurements together with the P2 receptor antagonists pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and suramin. Extracellular ATP (10 microM) almost completely depressed the thapsigargin (100 nM)-evoked [Ca(2+)](i) elevation mediated through store-operated Ca(2+) entry. The inhibitory effect of ATP was antagonized by PPADS with IC(50) of 0.7 microM. In the presence of PPADS at concentrations of more than 5 microM, the ATP-induced [Ca(2+)](i) elevation became sustained during the entire duration of the agonist application, although the magnitude of the sustained [Ca(2+)](i) elevation was reduced in a concentration-dependent manner by PPADS with an IC(50) of 200 microM. In contrast, the ATP-induced [Ca(2+)](i) elevation was blocked by suramin in a concentration range similar to that required to antagonize the inhibitory effect of ATP on the store-operated pathway. These results suggest that the [Ca(2+)](i) responses to extracellular ATP in rat brown adipocytes are mediated through the activation of at least two distinct P2 receptors exhibiting different sensitivities to PPADS but similar sensitivities to suramin. Extracellular ATP stimulates the PPADS-resistant P2 receptor to mobilize intracellular Ca(2+) stores, which is probably followed by the activation of store-operated Ca(2+) entry. Extracellular ATP, however, would inhibit this Ca(2+) entry process through the stimulation of the PPADS-sensitive P2-receptor, which may underlie the transient nature of [Ca(2+)](i) elevation in response to extracellular ATP.     

Involvement of prostaglandin E(2) derived from enteric glial cells in the action of bradykinin in cultured rat myenteric neurons

We characterized bradykinin (BK)-induced changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) and membrane potential in cultured rat myenteric neurons using ratiometric Ca(2+) imaging with fura-2 and the whole-cell patch-clamp technique, respectively. BK evoked a dose-dependent increase of [Ca(2+)]i that was abolished by HOE 140, a B2 receptor antagonist but not by [Lys-des-Arg(9)]-BK, a B1 receptor antagonist. [Lys-des-Arg(9)]-HOE140, a B1 receptor agonist, failed to cause a [Ca(2+)]i response. Double staining with antibodies against the B2 receptor together with PGP9.5 or S100 indicated that B2 receptors were expressed in neurons and glial cells. The BK-evoked [Ca(2+)]i increase was suppressed by indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and potentiated by prostaglandin E(2) (PGE(2)). The release of PGE(2) from cultured myenteric plexus cells was increased by BK. BK induced a large increase in [Ca(2+)]i in neurons when myenteric plexus cells were cultured at the high density but not at the low density, and caused a small increase in [Ca(2+)]i in neurons when proliferation of enteric glial cells was suppressed. BK evoked a slow and sustained depolarization in myenteric neurons, which was sensitive to indomethacin. These results indicated that BK caused a [Ca(2+)]i increase and depolarization in rat myenteric neurons through the activation of B2 receptors, which was partly associated with PGE(2) released from glial cells in response to BK. It is suggested that a neuron-glial interaction plays an important role in the effect of BK in the rat myenteric plexus.