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Intracellular Ca2+ Mobilization (intracellular + ca2+_mobilization)
Selected AbstractsCalcium-sensing receptor mediates phenylalanine-induced cholecystokinin secretion in enteroendocrine STC-1 cellsFEBS JOURNAL, Issue 18 2008Tohru Hira Intraluminal l -phenylalanine (Phe) stimulates cholecystokinin (CCK) secretion in vivo and in vitro. However, the cellular mechanism by which CCK-producing enteroendocrine cells sense Phe is unknown. The calcium-sensing receptor (CaR) can sense amino acids, and is expressed in the gastrointestinal tract. In the present study, we examined whether CaR functions as a receptor for Phe in CCK-producing enteroendocrine cells. CCK secretion and intracellular Ca2+ concentration in response to Phe were measured in the murine CCK-producing enteroendocrine cell line STC-1 at various extracellular Ca2+ concentrations or after treatment with a CaR antagonist. At more than 20 mm, Phe induced dose-dependent CCK secretion and intracellular Ca2+ mobilization in STC-1 cells. In the presence of 3.0 mm extracellular Ca2+, 10 and 20 mm Phe induced significantly higher CCK secretion than under normal conditions (1.2 mm extracellular Ca2+). Intracellular Ca2+ mobilization, induced by 10 or 20 mm Phe, was also enhanced by increasing extracellular Ca2+ concentrations. In addition, intracellular Ca2+ mobilization induced by addition of extracellular Ca2+ was augmented by the presence of Phe. These results closely match the known CaR properties. Treatment with a specific CaR antagonist (NPS2143) completely inhibited Phe-induced CCK secretion and the latter phase of intracellular Ca2+ mobilization. CaR mRNA expression was demonstrated by RT-PCR in STC-1 cells, as well as in other mouse tissues including the kidney, thyroid, stomach and intestine. In conclusion, CaR functions as a receptor for Phe, stimulating CCK secretion in enteroendocrine STC-1 cells. [source] Brain-derived neurotrophic factor induces long-lasting Ca2+ -activated K+ currents in rat visual cortex neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2002Yoshito Mizoguchi Abstract Brain-derived neurotrophic factor (BDNF) increases postsynaptic intracellular Ca2+ and modulates synaptic transmission in various types of neurons. Ca2+ -activated K+ currents, opened mainly by intracellular Ca2+ elevation, contribute to hyperpolarization following action potentials and modulate synaptic transmission. We asked whether BDNF induces Ca2+ -activated K+ currents by postsynaptic elevation of intracellular Ca2+ in acutely dissociated visual cortex neurons of rats. Currents were analysed using the nystatin-perforated patch clamp technique and imaging of intracellular Ca2+ mobilization with fura-2. At a holding potential of ,50 mV, BDNF application (20 ng/mL) for 1,2 min induced an outward current (IBDNF-OUT; 80.0 ± 29.0 pA) lasting for more than 90 min without attenuation in every neuron tested. K252a (200 nm), an inhibitor of Trk receptor tyrosine kinase, and U73122 (3 ,m), a specific phospholipase C (PLC)-, inhibitor, suppressed IBDNF-OUT completely. IBDNF-OUT was both charybdotoxin- (600 nm) and apamin- (300 nm) sensitive, suggesting that this current was carried by Ca2+ -activated K+ channels. BAPTA-AM (150 ,m) gradually suppressed IBDNF-OUT. Fura-2 imaging revealed that a brief application of BDNF elicited a long-lasting elevation of intracellular Ca2+. These results show that BDNF induces long-lasting Ca2+ -activated K+ currents by sustained intracellular Ca2+ elevation in rat visual cortex neurons. While BDNF, likely acting through the Trk B receptor, was necessary for the induction of long-lasting Ca2+ -activated K+ currents via intracellular Ca2+ elevation, BDNF was not necessary for the maintenance of this current. [source] Calcium-sensing receptor mediates phenylalanine-induced cholecystokinin secretion in enteroendocrine STC-1 cellsFEBS JOURNAL, Issue 18 2008Tohru Hira Intraluminal l -phenylalanine (Phe) stimulates cholecystokinin (CCK) secretion in vivo and in vitro. However, the cellular mechanism by which CCK-producing enteroendocrine cells sense Phe is unknown. The calcium-sensing receptor (CaR) can sense amino acids, and is expressed in the gastrointestinal tract. In the present study, we examined whether CaR functions as a receptor for Phe in CCK-producing enteroendocrine cells. CCK secretion and intracellular Ca2+ concentration in response to Phe were measured in the murine CCK-producing enteroendocrine cell line STC-1 at various extracellular Ca2+ concentrations or after treatment with a CaR antagonist. At more than 20 mm, Phe induced dose-dependent CCK secretion and intracellular Ca2+ mobilization in STC-1 cells. In the presence of 3.0 mm extracellular Ca2+, 10 and 20 mm Phe induced significantly higher CCK secretion than under normal conditions (1.2 mm extracellular Ca2+). Intracellular Ca2+ mobilization, induced by 10 or 20 mm Phe, was also enhanced by increasing extracellular Ca2+ concentrations. In addition, intracellular Ca2+ mobilization induced by addition of extracellular Ca2+ was augmented by the presence of Phe. These results closely match the known CaR properties. Treatment with a specific CaR antagonist (NPS2143) completely inhibited Phe-induced CCK secretion and the latter phase of intracellular Ca2+ mobilization. CaR mRNA expression was demonstrated by RT-PCR in STC-1 cells, as well as in other mouse tissues including the kidney, thyroid, stomach and intestine. In conclusion, CaR functions as a receptor for Phe, stimulating CCK secretion in enteroendocrine STC-1 cells. [source] Gq/11-induced intracellular calcium mobilization mediates Per2 acute induction in Rat-1 fibroblastsGENES TO CELLS, Issue 9 2006Naoyuki Takashima Phase resetting is one of the essential properties of circadian clocks that is required for the adjustment to a particular environment and the induction of Per1 and Per2 clock genes is believed to be a primary molecular event during this process. Although the intracellular signal transduction pathway underlying Per1 gene activation has been well characterized, the mechanisms that control Per2 up-regulation have not yet been elucidated. In our present study, we demonstrate that Gq/11 coupled receptors mediate serum-induced immediate rat Per2 (rPer2) transactivation in Rat-1 fibroblasts via intracellular Ca2+ mobilization. Stimulation of these cells with a high concentration of serum was found to rapidly increase the intracellular Ca2+ levels and strongly up-regulated rPer2 gene. rPer2 induction by serum stimulation was abrogated by intracellular Ca2+ chelation and depletion of intracellular Ca2+ store, which suggests that the calcium mobilization is necessary for the up-regulation of rPer2 gene. In addition, suppression of Gq/11 function was observed to inhibit both Ca2+ mobilization and rPer2 induction. Further, we demonstrated that endothelin-induced acute rPer2 transactivation via Gq/11-coupled endothelin receptors is also suppressed by a Gq/11 specific inhibitor. These findings together suggest that serum and endothelin utilize a common Gq/11-PLC mediated pathway for the transactivation of rPer2, which involves the mobilization of calcium from the intracellular calcium store. [source] Effects of prolactin on intracellular calcium concentration and cell proliferation in human glioma cellsGLIA, Issue 3 2002Thomas Ducret Abstract Prolactin (PRL) has several physiological effects on peripheral tissues and the brain. This hormone acts via its membrane receptor (PRL-R) to induce cell differentiation or proliferation. Using reverse transcription,polymerase chain reaction (RT-PCR) combined with Southern blot analysis, we detected PRL-R transcripts in a human glioma cell line (U87-MG) and in primary cultured human glioblastoma cells. These transcripts were deleted or not in their extracellular domains. We examined the effects of PRL on intracellular free Ca2+ concentration ([Ca2+]i) in these cells in order to improve our understanding of the PRL transduction mechanism, which is still poorly documented. [Ca2+]i was measured by microspectrofluorimetry using indo-1 as the Ca2+ fluorescent probe. Spatiotemporal aspects of PRL-induced Ca2+ signals were investigated using high-speed fluo-3 confocal imaging. We found that physiological concentrations (0.4,4 nM) of PRL-stimulated Ca2+ entry and intracellular Ca2+ mobilization via a tyrosine kinase,dependent mechanism. The two types of Ca2+ responses observed were distinguishable by their kinetics: one showing a slow (type I) and the other a fast (type II) increase in [Ca2+]i. The amplitude of PRL-induced Ca2+ increases may be sufficient to provoke several physiological responses, such as stimulating proliferation. Furthermore, PRL induced a dose-dependent increase in [3H]thymidine incorporation levels and in cellular growth and survival, detected by the MTT method. These data indicate that PRL induced mitogenesis of human glioma cells. GLIA 38:200,214, 2002. © 2002 Wiley-Liss, Inc. [source] Role of LOX-1 in monocyte adhesion-triggered redox, Akt/eNOS and Ca2+ signaling pathways in endothelial cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2009Nobuo Sakamoto This study was conducted to examine the role of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in monocyte adhesion-induced redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in endothelial cells (ECs). LOX-1 was blocked by an antibody-neutralizing LOX-1 TS92 or small interfering RNA. In cultured human aortic ECs, monocyte adhesion activated Rac1 and p47phox, and increased NADPH oxidase activity and reactive oxygen species (ROS) generation within 30,min and NF-,B phosphorylation within 1,h, resulting in redox-sensitive gene expression. Akt and eNOS phosphorylation was induced 15,min after adding monocytes and returned to control level after 30,min, whereas NO production was not altered by monocyte adhesion. Blockade of LOX-1 blunted the monocyte adhesion-triggered redox-sensitive signaling pathway and Akt/eNOS phosphorylation in ECs. Both endothelial intracellular Ca2+ mobilization and Ca2+ influx caused by monocyte attachment were markedly attenuated by pretreatment of ECs with TS92. This suggests that LOX-1 is involved in redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in monocyte adhesion to ECs independent of oxidized low-density lipoprotein (ox-LDL). Furthermore, blockade of Ca2+ inhibited monocyte adhesion-triggered Rac1 and p47phox activation and ROS generation in ECs, whereas Ca2+ signaling was suppressed by blockade of NADPH oxidase and ROS generation. Finally, TS92 blocked the monocyte adhesion to ECs stimulated with or without tumor necrosis factor-, or ox-LDL. We provide evidence that LOX-1 plays a role in redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in monocyte adhesion to ECs independent of the ox-LDL,LOX-1 axis. J. Cell. Physiol. 220: 706,715, 2009. © 2009 Wiley-Liss, Inc. [source] Sphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2004L. Formigli We have previously shown that sphingosine 1-phosphate (S1P) can induce intracellular Ca2+ mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca2+ -independent mechanisms of cell contraction have been the focus of numerous studies on Ca2+ sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho-dependent kinase mediate most of Ca2+ -independent contractile responses. In the present study, we examined the potential role of Rho/Rho-kinase cascade activation in S1P-induced C2C12 cell contraction. First, we showed that depletion of Ca2+, by pre-treatment with BAPTA, did not affect S1P-induced myoblastic contractility, whereas it abolished S1P-induced Ca2+ transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P-stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist-stimulation in cells depleted of Ca2+ and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y-27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with Gö6976 or rottlerin, specific inhibitors of PKC, and PKC,, respectively, failed to inhibit the agonist-induced myoblastic contraction. Single particle tracking of G-actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca2+ -independent/Rho-Rho kinase-dependent pathways may exert an important role in S1P-induced myoblastic cell contraction. J. Cell. Physiol. 198: 1,11, 2004© 2003 Wiley-Liss, Inc. [source] Recombinant human serotonin 5A receptors stably expressed in C6 glioma cells couple to multiple signal transduction pathwaysJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Mami Noda Abstract Human serotonin 5A (5-HT5A) receptors were stably expressed in undifferentiated C6 glioma. In 5-HT5A receptors-expressing cells, accumulation of cAMP by forskolin was inhibited by 5-HT as reported previously. Pertussis toxin-sensitive inhibition of ADP-ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine-sensitive Ca2+ mobilization. On the other hand, 5-HT-induced outward currents were observed using the patch-clamp technique in whole-cell configuration. The 5-HT-induced outward current was observed in 84% of the patched 5-HT5A receptor-expressing cells and was concentration-dependent. The 5-HT-induced current was inhibited when intracellular K+ was replaced with Cs+ but was not significantly inhibited by typical K+ channel blockers. The 5-HT-induced current was significantly attenuated by 1,2-bis(2-aminophenoxy)ethane- N,N,N,,N,-tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca2+ stores by application of caffeine or thapsigargin also blocked the 5-HT-induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5-HT-induced current. IP3 showed a transient increase after application of 5-HT in 5-HT5A receptor-expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP-ribosyl cyclase activity, 5-HT5A receptors regulate intracellular Ca2+ mobilization which is probably a result of the IP3-sensitive Ca2+ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function. [source] Role of Protein Kinases in the Prolactin-Induced Intracellular Calcium Rise in Chinese Hamster Ovary Cells Expressing the Prolactin ReceptorJOURNAL OF NEUROENDOCRINOLOGY, Issue 9 2000B. Sorin Abstract There is still only limited understanding of the early steps of prolactin signal transduction in target cells. It has been shown that prolactin actions are associated with cell protein phosphorylation, Ca2+ increases, and so on. However, the link between the activation of kinases and calcium influx or intracellular Ca2+ mobilization has not yet been clearly established. Chinese hamster ovary (CHO) cells, stably transfected with the long form of rabbit mammary gland prolactin receptor (PRL-R) cDNA were used for PRL-R signal transduction studies. Spectrofluorimetric techniques were used to measure intracellular calcium ([Ca2+]i) in cell populations with Indo1 as a calcium fluorescent probe. We demonstrate that, although protein kinase C activation (PMA or DiC8) caused a calcium influx in CHO cells, prolactin-induced PKC activation was not responsible for the early effect of prolactin on [Ca2+]i. Activation of protein kinase A (PKA) or protein kinase G did not modify [Ca2+]i and inhibition of PKA pathway did not affect the prolactin response. In the same way, phosphatidylinositol-3 kinaseinhibition had no effect on the prolactin-induced Ca2+ increase. On the other hand, tyrosine kinase inhibitors (herbimycin A, lavendustin A, and genistein) completely blocked the effect of prolactin on [Ca2+]i (influx and release). W7, a calmodulin-antagonist, and a specific inhibitor of calmodulin kinases (KN-62), only blocked prolactin-induced Ca2+ influx but had no significant effect on Ca2+ release. Using pharmacological agents, we present new data concerning the involvement of protein phosphorylations in the early effects of prolactin on ionic channels in CHO cells expressing the long form of PRL-R. Our results suggest that, at least in the very early steps of prolactin signal transduction, serine-threonine phosphorylation does not participate in the prolactin-induced calcium increase. On the other hand, tyrosine phosphorylation is a crucial, very early step, since it controls K+ channel activation, calcium influx, and intracellular calcium mobilization. Calmodulin acts later, since its inhibition only blocks the prolactin-induced Ca2+ influx. [source] Prostaglandin F2, upregulates interleukin-6 production in human gingival fibroblastsJOURNAL OF PERIODONTAL RESEARCH, Issue 2 2001Kazuyuki Noguchi Prostaglandin F2,(PGF2,) is a bioactive lipid mediator which has been suggested to be involved in the pathogenesis of periodontal disease. However, the roles of PGF2, in periodontal lesions are poorly understood. In the present study, we investigated the effect of PGF2, on interleukin (IL)-6 production in human gingival fibroblasts (HGF). PGF2,stimulated IL-6 production in a time- and concentration-dependent fashion. IL-1, and tumor necrosis factor ,(TNF,), proinflammatory cytokines, induced IL-6 production in a time-dependent manner, and PGF2,synergistically enhanced IL-6 production induced by IL-1, and TNF,. IL-6 mRNA was expressed in PGF2, -stimulated HGF, and PGF2, increased IL-6 mRNA levels induced by IL-1, and TNF,. Fluprostenol, a selective FP receptor agonist, could mimic PGF2, -induced IL-6 production. Since FP receptors are coupled to elevation of intracellular calcium and activation of protein kinase C (PKC), the mechanism of IL-6 production by PGF2, was investigated using TMB-8, an inhibitor of Ca2+ mobilization from intracellular stores, and calphostin C, an inhibitor of PKC. TMB-8 significantly suppressed PGF2, -induced IL-6 production, whereas calphostin C showed a stimulatory effect on PGF2, -induced IL-6 production. From these data, we suggest that PGF2, upregulates IL-6 production through FP receptors in HGF, that PGF2, synergistically enhances IL-6 production in IL-1,- and TNF,-stimulated HGF, and that PGF2, -induced IL-6 production may be dependent on intracellular Ca2+ mobilization and be downregulated by PKC activation. PGF2, may be involved in the pathogenesis of periodontal disease by enhancing IL-6 levels in periodontal lesions. [source] Mite serine protease activates protease-activated receptor-2 and induces cytokine release in human keratinocytesALLERGY, Issue 9 2009T. Kato Background:, House dust mites produce serine and cysteine proteases. Mite-derived proteases have been suggested to be involved in the pathogenesis of allergies; however, whether mite-derived serine protease activity can stimulate keratinocytes remains unknown. Methods:, We examined the activation of primary human keratinocytes by serine protease-rich extract of whole mite culture and compared with that by recombinant group 1 allergens (rDer f 1 and rDer p 1), which exclusively exhibit cysteine protease activity. Results:, Protease activity of whole mite culture extract (WCE), rDer f 1 and rDer p 1 induced the release of IL-8 and granulocyte-macrophage colony-stimulating factor. Protease activity of WCEs induced a significant upregulation of their mRNA expression but rDer f 1 had much less effect. Protease activity of the WCE stimulated intracellular Ca2+ mobilization but rDer f 1 and rDer p 1 did not. The mobilization induced by agonists for the human protease-activated receptor (PAR)-2, an agonist peptide or trypsin, was diminished by pre-incubation of keratinocytes with WCE. rDer f 1 inefficiently cleaved a synthetic N-terminal peptide of PAR-2 at different sites from trypsin, but the resultant peptides did not stimulate the release of interleukin-8. Conclusions:, The results suggest that mite-derived serine protease activity may contribute to the pathogenesis of atopic dermatitis by activating keratinocytes via PAR-2 activation but cysteine protease activity of Der f 1 and Der p 1 acts via another mechanism. [source] G protein-independent neuromodulatory action of adenosine on metabotropic glutamate signalling in mouse cerebellar Purkinje cellsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2007Toshihide Tabata Adenosine receptors (ARs) are G protein-coupled receptors (GPCRs) mediating the neuromodulatory actions of adenosine that influence emotional, cognitive, motor, and other functions in the central nervous system (CNS). Previous studies show complex formation between ARs and metabotropic glutamate receptors (mGluRs) in heterologous systems and close colocalization of ARs and mGluRs in several central neurons. Here we explored the possibility of intimate functional interplay between Gi/o protein-coupled A1 -subtype AR (A1R) and type-1 mGluR (mGluR1) naturally occurring in cerebellar Purkinje cells. Using a perforated-patch voltage-clamp technique, we found that both synthetic and endogenous agonists for A1R induced continuous depression of a mGluR1-coupled inward current. A1R agonists also depressed mGluR1-coupled intracellular Ca2+ mobilization monitored by fluorometry. A1R indeed mediated this depression because genetic depletion of A1R abolished it. Surprisingly, A1R agonist-induced depression persisted after blockade of Gi/o protein. The depression appeared to involve neither the cAMP-protein kinase A cascade downstream of the alpha subunits of Gi/o and Gs proteins, nor cytoplasmic Ca2+ that is suggested to be regulated by the beta-gamma subunit complex of Gi/o protein. Moreover, A1R did not appear to affect Gq protein which mediates the mGluR1-coupled responses. These findings suggest that A1R modulates mGluR1 signalling without the aid of the major G proteins. In this respect, the A1R-mediated depression of mGluR1 signalling shown here is clearly distinguished from the A1R-mediated neuronal responses described so far. These findings demonstrate a novel neuromodulatory action of adenosine in central neurons. [source] Cathelicidin LL-37 induces the generation of reactive oxygen species and release of human ,-defensins from neutrophilsBRITISH JOURNAL OF DERMATOLOGY, Issue 6 2007Y. Zheng Summary Background, Psoriasis is characterized by epidermal infiltration of neutrophils that destroy invading microorganisms via a potent antimicrobial arsenal of oxidants and antimicrobial agents. In contrast to atopic dermatitis, psoriasis exhibits low levels of skin infections due to the presence of antimicrobial agents, including cathelicidin LL-37. LL-37 kills a broad spectrum of microbes, and activates neutrophil chemotaxis. Objective, To determine whether or not LL-37 could regulate additional neutrophil functions such as production of cytokines/chemokines, reactive oxygen species and release of neutrophil antimicrobial peptides. Methods, Human peripheral blood neutrophils were used in this study. The production of interleukin (IL)-8 and release of ,-defensins were analysed by enzyme-linked immunosorbent assay, and real-time polymerase chain reaction (PCR) was used to quantify ,-defensin gene expression. Phosphorylation of mitogen-activated protein kinase (MAPK) was determined by Western blotting. The generation of reactive oxygen species was examined using flow cytometry, and intracellular Ca2+ mobilization was measured using a calcium assay kit. Results, LL-37 enhanced the production of IL-8 under the control of MAPK p38 and extracellular signal regulated kinase (ERK), as evidenced by the inhibitory effects of p38 and ERK1/2 inhibitors on LL-37-mediated IL-8 production. Furthermore, LL-37 induced phosphorylation of p38 and ERK. We also revealed that LL-37 stimulated the generation of reactive oxygen species dose- and time-dependently, most probably via NADPH oxidase activation and intracellular Ca2+ mobilization. Finally, LL-37 induced both mRNA expression and protein release of ,-defensins, known as human neutrophil peptide 1,3. Conclusion, Taken together, we suggest that in addition to its microbicidal properties, LL-37 may contribute to innate immunity by enhancing neutrophil host defence functions at inflammation and/or infection sites. [source] SALIVATION TRIGGERED BY PILOCARPINE INVOLVES AQUAPORIN-5 IN NORMAL RATS BUT NOT IN IRRADIATED RATSCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 5-6 2009Tetsuya Asari SUMMARY 1Using rats, we examined the muscarinic receptor subtype mediating pilocarpine-induced parotid salivary secretion and the contributions of ion transporter systems (effluxes of K+ and Cl - ) and aquaporin-5 (AQP5) translocation to this response in parotid glands in irradiated-induced xerostomia. 2Salivary secretion was significantly lower in irradiated compared with sham-irradiated (normal) rats. In xerostomia rats, 0.4 and 0.8 mg/kg pilocarpine significantly increased parotid salivary secretion, although the salivary volume was still significantly less than in normal rats after the same dose of pilocarpine. 3Pirenzepine (1 × 10,6 to 1 × 10,1 mol/L), AF-DX 116 (3 × 10,6 to 3 × 10,2 mol/L) and N -2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP; 1 × 10,8 to 1 × 10,2 mol/L) dose-dependently displaced radioligand binding to M1, M2 and M3 receptors, respectively, in parotid membranes from both normal and irradiated rats. In each group of rats, 4-DAMP had the highest binding affinity. Pretreatment with 4-DAMP or pirenzepine dose-dependently inhibited pilocarpine-induced parotid secretion in both normal and irradiated rats, with 4-DAMP being markedly more potent than pirenzepine. 4Normal and irradiated-rat parotid cells did not differ significantly in terms of pilocarpine-induced changes in [Ca2+]i, [K+]i and [Cl - ]i. Pilocarpine markedly increased the amount of AQP5 in the apical plasma membrane of parotid cells isolated from normal but not irradiated rats. 5Thus, pilocarpine induces parotid salivary secretion mainly via the M3 receptor subtype in both irradiated and normal rats. The reduction in this pilocarpine-induced secretion seen in irradiated rats is due not to disturbances of intracellular Ca2+ mobilization or ion transporter systems, but rather to a disturbance of AQP5 translocation, which may be involved in the pathogenesis of X-ray irradiation-induced xerostomia. [source] | |||||||||||||