Identification of phospholipase C β downstream effect on transient receptor potential canonical 1/4, transient receptor potential canonical 1/5 channels

Gα(q)-coupled receptor stimulation was implied in the activation process of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heterotetrameric channels. The inactivation occurs due to phosphatidylinositol 4,5-biphosphate (PI(4,5)P₂) depletion. When PI(4,5)P₂ depletion was induced by muscarinic stimulation or inositol polyphosphate 5-phosphatase (Inp54p), however, the inactivation by muscarinic stimulation was greater compared to that by Inp54p. The aim of this study was to investigate the complete inactivation mechanism of the heteromeric channels upon Gα(q)-phospholipase C β (Gα(q)-PLCβ) activation. We evaluated the activity of heteromeric channels with electrophysiological recording in HEK293 cells expressing TRPC channels. TRPC1/4 and TRPC1/5 heteromers undergo further inhibition in PLCβ activation and calcium/protein kinase C (PKC) signaling. Nevertheless, the key factors differ. For TRPC1/4, the inactivation process was facilitated by Ca²⁺ release from the endoplasmic reticulum, and for TRPC1/5, activation of PKC was concerned mostly. We conclude that the subsequent increase in cytoplasmic Ca²⁺ due to Ca²⁺ release from the endoplasmic reticulum and activation of PKC resulted in a second phase of channel inhibition following PI(4,5)P₂ depletion.

The Inhibitory Mechanism on Acetylcholine-Induced Contraction of Bladder Smooth Muscle in the Streptozotocin-Induced Diabetic Rat

Most diabetic patients experience diabetic mellitus (DM) urinary bladder dysfunction. A number of studies evaluate bladder smooth muscle contraction in DM. In this study, we evaluated the change of bladder smooth muscle contraction between normal rats and DM rats. Furthermore, we used pharmacological inhibitors to determine the differences in the signaling pathways between normal and DM rats. Rats in the DM group received an intraperitoneal injection of 65 mg/kg streptozotocin and measured blood glucose level after 14 days to confirm DM. Bladder smooth muscle contraction was induced using acetylcholine (ACh, 10⁻⁴ M). The materials such as, atropine (a muscarinic receptor antagonist), U73122 (a phospholipase C inhibitor), DPCPX (an adenosine A1 receptor antagonist), udenafil (a PDE5 inhibitor), prazosin (an α₁-receptor antagonist), papaverine (a smooth muscle relaxant), verapamil (a calcium channel blocker), and chelerythrine (a protein kinase C inhibitor) were pre-treated in bladder smooth muscle. We found that the DM rats had lower bladder smooth muscle contractility than normal rats. When prazosin, udenafil, verapamil, and U73122 were pre-treated, there were significant differences between normal and DM rats. Taken together, it was concluded that the change of intracellular Ca²⁺ release mediated by PLC/IP3 and PDE5 activity were responsible for decreased bladder smooth muscle contractility in DM rats.

The change of signaling pathway on the electrical stimulated contraction in streptozotocin-induced bladder dysfunction of rats

Bladder dysfunction is a common complication of diabetes mellitus (DM). However, there have been a few studies evaluating bladder smooth muscle contraction in DM in the presence of pharmacological inhibitors. In the present study, we compared the contractility of bladder smooth muscle from normal rats and DM rats. Furthermore, we utilized pharmacological inhibitors to delineate the mechanisms underlying bladder muscle differences between normal and DM rats. DM was established in 14 days after using a single injection of streptozotocin (65 mg/kg, intraperitoneal) in Sprague-Dawley rats. Bladder smooth muscle contraction was induced electrically using electrical field stimulation consisting of pulse trains at an amplitude of 40 V and pulse duration of 1 ms at frequencies of 2–10 Hz. In this study, the pharmacological inhibitors atropine (muscarinic receptor antagonist), U73122 (phospholipase C inhibitor), DPCPX (adenosine A₁ receptor antagonist), udenafil (PDE5 inhibitor), prazosin (α₁-receptor antagonist), verapamil (calcium channel blocker), and chelerythrine (protein kinase C inhibitor) were used to pretreat bladder smooth muscles. It was found that the contractility of bladder smooth muscles from DM rats was lower than that of normal rats. In addition, there were significant differences in percent change of contractility between normal and DM rats following pretreatment with prazosin, udenafil, verapamil, and U73122. In conclusion, we suggest that the decreased bladder muscle contractility in DM rats was a result of perturbations in PLC/IP₃-mediated intracellular Ca²⁺ release and PDE5 activity.

Effect of Sphingosine-1-Phosphate on Intracellular Free Ca2+ in Cat Esophageal Smooth Muscle Cells

A comprehensive collection of proteins senses local changes in intracellular Ca²⁺ concentrations ([Ca²⁺](i) and transduces these signals into responses to agonists. In the present study, we examined the effect of sphingosine-1-phosphate (S1P) on modulation of intracellular Ca²⁺ concentrations in cat esophageal smooth muscle cells. To measure [Ca²⁺](i) levels in cat esophageal smooth muscle cells, we used a fluorescence microscopy with the Fura-2 loading method. S1P produced a concentration-dependent increase in [Ca²⁺](i) in the cells. Pretreatment with EGTA, an extracellular Ca²⁺ chelator, decreased the S1P-induced increase in [Ca²⁺](i), and an L-type Ca²⁺-channel blocker, nimodipine, decreased the effect of S1P. This indicates that Ca²⁺ influx may be required for muscle contraction by S1P. When stimulated with thapsigargin, an intracellular calcium chelator, or 2-Aminoethoxydiphenyl borate (2-APB), an InsP₃ receptor blocker, the S1P-evoked increase in [Ca²⁺](i) was significantly decreased. Treatment with pertussis toxin (PTX), an inhibitor of G(i)-protein, suppressed the increase in [Ca²⁺](i) evoked by S1P. These results suggest that the S1P-induced increase in [Ca²⁺](i) in cat esophageal smooth muscle cells occurs upon the activation of phospholipase C and subsequent release of Ca²⁺ from the InsP₃-sensitive Ca²⁺ pool in the sarcoplasmic reticulum. These results suggest that S1P utilized extracellular Ca²⁺ via the L type Ca²⁺ channel, which was dependent on activation of the S1P₄ receptor coupled to PTX-sensitive G(i) protein, via phospholipase C-mediated Ca²⁺ release from the InsP₃-sensitive Ca²⁺ pool in cat esophageal smooth muscle cells.

PLCE1 Promotes the Invasion and Migration of Esophageal Cancer Cells by Up-Regulating the PKCα/NF-κB Pathway

PURPOSE: To investigate the effect and mechanism of phospholipase C epsilon gene 1 (PLCE1) expression on esophageal cancer cell lines. MATERIALS AND METHODS: The esophageal carcinoma cell lines Eca109 and EC9706 and normal esophageal epithelial cell line HEEC were cultured. The expression of PLCE1, protein kinase C alpha (PKCα), and nuclear factor kappa B (NF-κB) p50/p65 homodimer in cells were comparatively analyzed. The esophageal cancer cells were divided into si-PLCE1, control siRNA (scramble), and mock groups that were transfected with specific siRNA for PLCE1, control siRNA, and blank controls, respectively. Expression of PLCE1, PKCα, p50, and p65 was detected by Western blotting. Transwell assay was used to detect migration and invasion of Eca109 and EC9706 cells. RESULTS: Compared with HEEC, the expression of PLCE1, PKCα, p50, and p65 was increased in Eca109 and EC9706 cells. The expression of PLCE1 was positively correlated with the expression of PKCα and p50 (PKCα: r=0.6328, p=0.032; p50: r=0.6754, p=0.041). PKCα expression had a positive correlation with the expression of p50 and p65 (p50: r=0.9127, p=0.000; p65: r=0.9256, p=0.000). Down-regulation of PLCE1 significantly decreased the expression of PKCα and NF-κB-related proteins (p65: p=0.002, p=0.004; p50: p=0.005, p=0.009) and inhibited the migration and invasion of Eca109 and EC9706 cells. CONCLUSION: PLCE1 activated NF-κB signaling by up-regulating PKCα, which could promote invasion and migration of esophageal cancer cells.

Relationship between potential sperm factors involved in oocyte activation and sperm DNA fragmentation with intra-cytoplasmic sperm injection clinical outcomes

Objective: The present study aimed to simultaneously evaluate the association between expression of three potential factors [post-acrosomal sheath WW domain-binding protein [PAWP], phospholipase Czeta [PLCzeta], and truncated form of the kit receptor [TR-KIT]] as candidates of oocyte activation with fertilization rate and early embryonic development

Materials and Methods: In this experimental study, semen samples were collected from 35 intra-cytoplasmic sperm injection [ICSI] candidates and analyzed according to World Health Organization criteria [2010]. Each sample was divided into two parts. The first part was processed for insemination by density-gradient centrifugation [DGC] and the second part was prepared for assessment of sperm morphology [Papanicolaou staining], DNA fragmentation [transferase dUTP nick end labeling [TUNEL]], and three Sperm-borne oocyte-activating factor [s] [SOAFs]-PLCzeta, PAWP, and TR-KIT

Results: Significant positive correlations existed between the percentages of PLCzeta, PAWP, and TR-KIT with fertilization rate. In addition, significant negative correlations existed between the percentage of DNA fragmentation with the percentages of PLCzeta and PAWP. We did not find a relationship between percentages of PLCzeta, PAWP, and TR-KIT with embryo quality and pregnancy rate [P>0.05]. There was a significant negative correlation between percentage of DNA fragmentation with fertilization and embryo quality

Conclusion: Oocyte activation was associated with the studied sperm factors [PAWP, PLCzeta, and TR-KIT]. These factors might hold the potential to be considered as diagnostic factors in the assessment of semen samples to evaluate their potential to induce oocyte activation. In addition, we observed a significant association between DNA fragmentation with fertilization, as well as embryo quality and expression of PAWP and PLCzeta, which indicated that men with high degrees of DNA fragmentation might require artificial oocyte activation. Whether such action should take place, and its cost and benefits should be evaluated in the future

Gintonin, an exogenous ginseng-derived LPA receptor ligand, promotes corneal wound healing

Ginseng gintonin is an exogenous ligand of lysophosphatidic acid (LPA) receptors. Accumulating evidence shows LPA helps in rapid recovery of corneal damage. The aim of this study was to evaluate the therapeutic efficacy of gintonin in a rabbit model of corneal damage. We investigated the signal transduction pathway of gintonin in human corneal epithelium (HCE) cells to elucidate the underlying molecular mechanism. We next evaluated the therapeutic effects of gintonin, using a rabbit model of corneal damage, by undertaking histochemical analysis. Treatment of gintonin to HCE cells induced transient increases of [Ca²⁺](i) in concentration-dependent and reversible manners. Gintonin-mediated mobilization of [Ca²⁺](i) was attenuated by LPA1/3 receptor antagonist Ki16425, phospholipase C inhibitor U73122, inositol 1,4,5-triphosphate receptor antagonist 2-APB, and intracellular Ca²⁺ chelator BAPTA-AM. Gintonin facilitated in vitro wound healing in a concentration-dependent manner. When applied as an eye-drop to rabbits with corneal damage, gintonin rapidly promoted recovery. Histochemical analysis showed gintonin decreased corneal apoptosis and increased corneal cell proliferation. We demonstrated that LPA receptor activation by gintonin is linked to in vitro and in vivo therapeutic effects against corneal damage. Gintonin can be applied as a clinical agent for the rapid healing of corneal damage.

Glucosylsphingosine Activates Serotonin Receptor 2a and 2b: Implication of a Novel Itch Signaling Pathway

Recent reports claimed that glucosylsphingosine (GS) is highly accumulated and specifically evoking itch-scratch responses in the skins of atopic dermatitis (AD) patients. However, it was unclear how GS can trigger itch-scratch responses, since there were no known molecular singling pathways revealed yet. In the present study, it was verified for the first time that GS can activate mouse serotonin receptor 2a (mHtr2a) and 2b (mHtr2b), but not 2c (mHtr2c) that are expressed in HEK293T cells. Specifically, effects of GS on all mouse serotonin receptor 2 subfamily were evaluated by calcium imaging techniques. The GS-induced intracellular calcium increase was dose-dependent, and antagonists such as ketanserin (Htr2a antagonist) and RS-127445 (Htr2b antagonist) significantly blocked the GS-induced responses. Moreover, the proposed GS-induced responses appear to be mediated by phospholipase C (PLC), since pretreatment of a PLC inhibitor U-73122 abolished the GS-induced responses. Additionally, the GS-induced calcium influx is probably mediated by endogenous TRPC ion channels in HEK293T cells, since pretreatment of SKF-96365, an inhibitor for TRPC, significantly suppressed GS-induced response. In conclusion, the present study revealed for the first time that GS can stimulate mHtr2a and mHtr2b to induce calcium influx, by utilizing PLC-dependent pathway afterwards. Considering that GS is regarded as a pruritogen in AD, the present study implicates a novel GS-induced itch signaling pathway.

Sarcoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2) reduces the migratory capacity of CCL21-treated monocyte-derived dendritic cells

The migration of dendritic cells (DCs) to secondary lymphoid organs depends on chemoattraction through the interaction of the chemokine receptors with chemokines. However, the mechanism of how lymphoid chemokines attract DCs to lymphoid organs remains unclear. Here, we demonstrate the mechanism of DC migration in response to the lymphoid chemokine CCL21. CCL21-mediated DC migration is controlled by the regulation of sarcoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2) expression rather than through the activation of mitogen-activated protein kinases CCL21-exposed mature DCs (mDCs) exhibited decreased SERCA2 expression but not decreased phospholamban (PLB) or Hax-1 expression, which are known to be SERCA2-interacting proteins. In addition, CCL21 did not affect the mRNA levels of SERCA2 or its interacting protein Hax-1. Interestingly, SERCA2 expression was inversely related to DC migration in response to chemokine stimulation. The migratory capacity of CCL21-treated mDCs was decreased by the phospholipase C inhibitor U73122 and by the protein kinase C inhibitor BAPTA-AM. The migratory capacities of mDCs were increased in response to SERCA2 siRNA expression but were decreased by SERCA2 overexpression. In addition, DCs treated with a SERCA2-specific inhibitor (cyclopiazonic acid) had significantly increased migratory capacities as mDCs regardless of SERCA2 expression. Moreover, SERCA2 expression was dependent on DC maturation induced by cytokines or Toll-like receptor agonists. Therefore, the migratory capacities differed in differentially matured DCs. Taken together, these results suggest that SERCA2 contributes to the migration of CCL21-activated DCs as an important feature of the adaptive immune response and provide novel insights regarding the role of SERCA2 in DC functions.

Effect of Chaiqin Chengqi Decoction on cholecystokinin receptor 1-mediated signal transduction of pancreatic acinar cells in acute necrotizing pancreatitis rats / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of Chaiqin Chengqi Decoction (,CQCQD) on cholecystokinin receptor 1 (CCKR1)-mediated signal transduction of pancreatic acinar cell in rats with acute necrotic pancreatitis (ANP).</p><p><b>METHODS</b>Twenty-seven Sprague-Dawley rats were randomized into three groups: the control group, the ANP group, and the CQCQD group (9 in each group). ANP rats were induced by two intraperitoneal injections of 8% L-arginine (pH=7.0, 4.4 g/kg) over a 2-h period. Rats were treated with 1.5 mL/100 g body weight of CQCQD (CQCQD group) or physiological saline (control and ANP groups) at 2 h interval. And 6 h after induction, pancreatic tissues were collected for histopathological examination. Pancreatic acinar cells were isolated for determination of CCKR1 mRNA and protein expression, phospholipase C (PLC) and inositol-1,4,5-triphosphate (IP3), and determination of fluorescence intensity (FI) as a measure of intracellular calcium ion concentration [Ca(2+)]i.</p><p><b>RESULTS</b>The pancreatic histopathological score (6.2 ± 1.1) and the levels of PLC (1,187.2 ± 228.2 μg/mL) and IP3 (872.2 ± 88.4 μg/mL) of acinar cells in the ANP group were higher than those in the control (2.8 ± 0.4, 682.5 ± 121.8 μg/mL, 518.4 ± 115.8 μg/mL) and the CQCQD (3.8 ± 0.8, 905.3 ± 78.5 μg/mL, 611.0 ± 42.5 μg/mL) groups (P<0.05). [Ca(2+)]i FI for the ANP group (34.8±27.0) was higher than that in the control (5.1 ± 2.2) and CQCQD (12.6 ± 2.5) groups (P<0.05). The expression of pancreatic acinar cell CCKR1 mRNA in the ANP group was up-regulated (expression ratio=1.761; P=0.024) compared with the control group. The expression of pancreatic acinar cell CCKR1 mRNA in the CQCQD group was down-regulated (expression ratio=0.311; P=0.035) compared with the ANP group. The ratio of gray values of the CCKR1 and β-actin in the ANP group (1.43 ± 0.17) was higher than those in the control (0.70 ± 0.15) and CQCQD (0.79 ± 0.11) groups (P<0.05).</p><p><b>CONCLUSIONS</b>Pancreatic acinar cell calcium overload of ANP induced by L-arginine was related to the up-regulated expressions of pancreatic acinar cell CCKR1 mRNA and protein. CQCQD can down-regulate expressions of pancreatic acinar cell CCKR1 mRNA and protein to reduce the PLC and IP3 of pancreatic acinar cells, relieving the calcium overload and reducing the pathological changes in rats with ANP.</p>

Activation of phospholipase C-independent protein kinase C signaling pathway of parathyroid hormone enhances CITED1 expression in mouse osteoblasts / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the functions of phospholipase C (PLC)-independent protein kinase C signaling pathway (PTH/nonPLC/PKC) of parathyroid hormone (PTH) and its role in bone metabolism.</p><p><b>METHODS</b>Osteoblasts isolated from the calvaria of 2- or 3-day-old C57BL mice, identified by alkaline phosphatase staining and Alizarin red staining, were treated for 4 h with 100 nmol/L [Gly(1), Arg(19)]hPTH(1-28) plus 10 nmol/L RP-cAMP, 10 nmol/L [Gly(1), Arg(19)]hPTH(1-34) plus 10 nmol/L RP-cAMP , 10 nmol/L PTH(1-34), or and 0.1% trifluoroacetic acid (TFA). The total RNA was then isolated for screening differentially expressed genes related to PTH/nonPLC/PKC pathway using Affymetrix mouse 12x135K gene expression profile microarray, and the identified genes were confirmed by real-time quantitative PCR. MC3T3-E1 cells treated with [Gly(1), Arg(19)]hPTH(1-28)+RP-cAMP, [Gly(1), Arg(19)]hPTH(1-34)+RP-cAMP, [Gly(1), Arg(19)]hPTH(1-34)+ RP-cAMP +100 nmol/L Go6983, or 0.1% TFA were also examined for GR(1-28)- or GR(1-34)-mediated gene expression changes using real-time quantitative PCR.</p><p><b>RESULTS</b>Alizarin red staining visualized red mineralized nodules in the osteoblasts at 28 days of culture. According to the genechip results, we selected 56 target genes related to PTH/nonPLC/PKC pathway, among which CITED1 showed higher expressions in [Gly(1), Arg(19)]hPTH(1-34)+ RP-cAMP group than in both the control group and [Gly(1), Arg(19)]hPTH(1-28)+RP-cAMP group (P<0.05), and its expression was the highest in PTH(1-34) group (P<0.05). RT-PCR of MC3T3-E1 cells yielded consist results with those in the primary osteoblasts, and the cells treated with Go6983 (a PKC inhibitor) did not show GR(1-28)- or GR(1-34)-mediated differential expression of CITED1.</p><p><b>CONCLUSION</b>The activation of PLC-independent protein kinase C signaling pathway of PTH enhances the expression of CITED1 in mouse osteoblasts to mediate the effect of PTH on bone metabolism, and this pathway is not dependent on the activation of PLC or PKA signaling.</p>

House Dust Mite Extract Induces PLC/IP3-dependent Ca2+ Signaling and IL-8 Expression in Human Gingival Epithelial Cells

The gingival epithelium of the oral cavity is constantly exposed to exogenous stimuli such as bacterial toxins, allergens, and thermal changes. These exogenous stimuli are resisted by innate host defense in gingival epithelial cells. However, it is unclear exactly how the exogenous stimuli affect detrimentally on the human gingival epithelial cells. Here, we investigated whether the allergen, such as house dust mite (HDM) extract, is linked to Ca2+ signaling and proinflammatory cytokine expression in primary cultured human gingival epithelial cells. HDM extract induced an increase in intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. Extracellular Ca2+ depletion did not affected on the HDM extract-induced increase in [Ca2+]i. The HDM extract-induced increase in [Ca2+]i was abolished by the treatment with U73122 and 2-APB, which are inhibitors of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) receptor. Moreover, HDM extract induced the mRNA expression of pro-inflammatory cytokine, interleukin (IL)-8. These results suggest that HDM extract triggers PLC/IP3-dependent Ca2+ signaling and IL-8 mRNA expression in primary cultured human gingival epithelial cells.

Relationship between phospholipase C zeta immunoreactivity and DNA fragmentation and oxidation in human sperm

OBJECTIVE: The study aimed to evaluate the feasibility and reproducibility of measuring phospholipase C zeta (PLCzeta) using immunostaining in human sperm and to investigate the relationship between PLCzeta immunoreactivity and DNA fragmentation and oxidation in human sperm. METHODS: Semen samples were obtained from participants (n=44) and processed by the conventional swim-up method. Sperm concentration, motility, normal form by strict morphology, DNA fragmentation index assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling method and immunofluorescent expression for 8-hydroxy-2'-deoxyguanosine (8-OHdG) and PLCzeta were assessed. RESULTS: When duplicate PLCzeta tests were performed on two sperm samples from each of the 44 participants, similar results were obtained (74.1+/-9.4% vs. 75.4+/-9.7%). Two measurements of PLCzeta were found to be highly correlated with each other (r=0.759, P<0.001). Immunoreactivity of PLCzeta was not associated with donor's age, sperm concentration, motility, and the percentage of normal form as well as DNA fragmentation index. However, immunoreactivity of PLCzeta showed a significant negative relationship with 8-OHdG immunoreactivity (r=-0.404, P=0.009). CONCLUSION: Measurement of PLCzeta by immunostaining is feasible and reproducible. Lower expression of PLCzeta in human sperm may be associated with higher sperm DNA oxidation status.

Peptidoglycan Induces the Production of Interleukin-8 via Calcium Signaling in Human Gingival Epithelium

The etiology of periodontal disease is multifactorial. Exogenous stimuli such as bacterial pathogens can interact with toll-like receptors to activate intracellular calcium signaling in gingival epithelium and other tissues. The triggering of calcium signaling induces the secretion of pro-inflammatory cytokines such as interleukin-8 as part of the inflammatory response; however, the exact mechanism of calcium signaling induced by bacterial toxins when gingival epithelial cells are exposed to pathogens is unclear. Here, we investigate calcium signaling induced by bacteria and expression of inflammatory cytokines in human gingival epithelial cells. We found that peptidoglycan, a constituent of gram-positive bacteria and an agonist of toll-like receptor 2, increases intracellular calcium in a concentration-dependent manner. Peptidoglycan-induced calcium signaling was abolished by treatment with blockers of phospholipase C (U73122), inositol 1,4,5-trisphosphate receptors, indicating the release of calcium from intracellular calcium stores. Peptidoglycan-mediated interleukin-8 expression was blocked by U73122 and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester). Moreover, interleukin-8 expression was induced by thapsigargin, a selective inhibitor of the sarco/endoplasmic reticulum calcium ATPase, when thapsigargin was treated alone or co-treated with peptidoglycan. These results suggest that the gram-positive bacterial toxin peptidoglycan induces calcium signaling via the phospholipase C/inositol 1,4,5-trisphosphate pathway, and that increased interleukin-8 expression is mediated by intracellular calcium levels in human gingival epithelial cells.

The Inhibitory Mechanism of Gentamicin on Electrical Field Stimulation Response in Rat Bladder Smooth Muscle

To see the inhibitory mechanism of gentamicin in response to electrical field stimulation (EFS) using the rat bladder smooth muscle, atropine or guanethidine was treated but had no effect. Methylsergide, a non-selective 5-HT1, 5-HT2 receptor antagonist was also treated but had on effect. Kinase inhibitors, such as chelerythrine (PKC inhibitor), ML-9 (MLCK inhibitor), or Y27632 (rho kinase inhibitor) were pretreated before gentamicin treatment, but did not have effect. For U73122, a phospholipase C (PLC) inhibitor however, the inhibitory effect to gentamicin was significantly attenuated in all frequencies given by the EFS. Therefore gentamicin induced inhibitory effect on EFS response in rat bladder smooth muscle was not mediated by the activation of adrenergic, cholinergic, or serotonergic receptor. The inhibition of gentamicin might be mediated through the PLC dependent pathway, but not through the PKC, MLCK or rho kinase dependent pathway.

Wnt5a stimulates chemotactic migration and chemokine production in human neutrophils

Wnt5a is a ligand that activates the noncanonical Wnt signaling pathways (beta-catenin-independent pathways). Human neutrophils expressed several Wnt5a receptors, such as Frizzled 2, 5 and 8. Stimulation of human neutrophils with Wnt5a caused chemotactic migration and the production of two important chemokines, CXCL8 and CCL2. CCL2 production by Wnt5a was mediated by a pertussis toxin-sensitive G-protein-dependent pathway. Wnt5a also stimulated the phosphorylation of three mitogen-activated protein kinases (MAPKs: ERK, p38 MAPK and JNK) and Akt. Inhibition of ERK, p38 MAPK or JNK by specific inhibitors induced a dramatic reduction in Wnt5a-induced CCL2 production. Supernatant collected from lipopolysaccharide-stimulated macrophages induced neutrophil chemotaxis, which was significantly inhibited by anti-Wnt5a antibody. Our results suggested that Wnt5a may contribute to neutrophil recruitment, mediating the inflammation response.

Effects of Histamine on Cultured Interstitial Cells of Cajal in Murine Small Intestine

Interstitial cells of Cajal (ICCs) are the pacemaker cells in the gastrointestinal tract, and histamine is known to regulate neuronal activity, control vascular tone, alter endothelial permeability, and modulate gastric acid secretion. However, the action mechanisms of histamine in mouse small intestinal ICCs have not been previously investigated, and thus, in the present study, we investigated the effects of histamine on mouse small intestinal ICCs, and sought to identify the receptors involved. Enzymatic digestions were used to dissociate ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record potentials (in current clamp mode) from cultured ICCs. Histamine was found to depolarize resting membrane potentials concentration dependently, and whereas 2-PEA (a selective H1 receptor agonist) induced membrane depolarizations, Dimaprit (a selective H2-agonist), R-alpha-methylhistamine (R-alpha-MeHa; a selective H3-agonist), and 4-methylhistamine (4-MH; a selective H4-agonist) did not. Pretreatment with Ca(2+)-free solution or thapsigargin (a Ca(2+)-ATPase inhibitor in endoplasmic reticulum) abolished the generation of pacemaker potentials and suppressed histamine-induced membrane depolarization. Furthermore, treatments with U-73122 (a phospholipase C inhibitor) or 5-fluoro-2-indolyl des-chlorohalopemide (FIPI; a phospholipase D inhibitor) blocked histamine-induced membrane depolarizations in ICCs. On the other hand, KT5720 (a protein kinase A inhibitor) did not block histamine-induced membrane depolarization. These results suggest that histamine modulates pacemaker potentials through H1 receptor-mediated pathways via external Ca2+ influx and Ca2+ release from internal stores in a PLC and PLD dependent manner.

Signaling Pathway of Lysophosphatidic Acid-Induced Contraction in Feline Esophageal Smooth Muscle Cells

Lysolipids such as LPA, S1P and SPC have diverse biological activities including cell proliferation, differentiation, and migration. We investigated signaling pathways of LPA-induced contraction in feline esophageal smooth muscle cells. We used freshly isolated smooth muscle cells and permeabilized cells from cat esophagus to measure the length of cells. Maximal contraction occurred at 10(-6) M and the response peaked at 30s. To identify LPA receptor subtypes in cells, western blot analysis was performed with antibodies to LPA receptor subtypes. LPA1 and LPA3 receptor were detected at 50 kDa and 44 kDa. LPA-induced contraction was almost completely blocked by LPA receptor (1/3) antagonist KI16425. Pertussis toxin (PTX) inhibited the contraction induced by LPA, suggesting that the contraction is mediated by a PTX-sensitive G protein. Phospholipase C (PLC) inhibitors U73122 and neomycin, and protein kinase C (PKC) inhibitor GF109203X also reduced the contraction. The PKC-mediated contraction may be isozyme-specific since only PKCepsilon antibody inhibited the contraction. MEK inhibitor PD98059 and JNK inhibitor SP600125 blocked the contraction. However, there is no synergistic effect of PKC and MAPK on the LPA-induced contraction. In addition, RhoA inhibitor C3 exoenzyme and ROCK inhibitor Y27632 significantly, but not completely, reduced the contraction. The present study demonstrated that LPA-induced contraction seems to be mediated by LPA receptors (1/3), coupled to PTX-sensitive G protein, resulting in activation of PLC, PKC-epsilon pathway, which subsequently mediates activation of ERK and JNK. The data also suggest that RhoA/ROCK are involved in the LPA-induced contraction.

Role of phospholipase C in cytoskeleton rearrangements of dendritic cells invaded by Mycobacterium tuberculosis / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the role of phospholipase C(PLC) in cytoskeleton rearrangement of mouse dendritic cells invaded by Mycobacterium tuberculosis.</p><p><b>METHODS</b>Mouse dendritic DC2.4 cells were co-cultured with Mycobacterium tuberculosis H37Rv. F-actin of DC2.4 cells were strained with phalloidin-TRITC, the microtubule was stained with anti-β-tubulin monoclonal antibody and FITC-conjugated AffiniPure anti-mouse IgG. The changes of cytoskeleton in DC2.4 cells induced by Mycobacterium tuberculosis H37Rv were determined by fluorescence microscopy and the rates of F-actin rearrangements were calculated. The expressions of PLC in cytoplasm and cytomembrane of DC2.4 cells were measured by ELISA. DC2.4 cells were pretreated with PLC inhibitor U73122, then F-actin rearrangements induced by invasion of Mycobacterium tuberculosis were observed.</p><p><b>RESULTS</b>Bacterial invasion was observed while DC2.4 cells were co-incubated with Mycobacterium tuberculosis H37Rv for 2 h. The rates of invasion were (26.1 ± 4.5)%, (39.9 ± 5.6)%, (51.2 ±5.9)%, (57.9 ± 6.1)% and (63.9 ± 6.8)% at 4, 6, 8, 10 and 12 h of co-culture, respectively; while those were (13.6 ± 3.1)%, (14.2 ± 3.9)%, (15.1 ± 4.3)%, (16.8 ± 4.0)% and (18.3 ± 5.2)% after blocked by PLC, respectively. The rates of the F-actin rearrangements at 2, 4, 6, 8, 10 and 12 h after DC2.4 cells were invaded by H37Rv were (26.9 ± 1.5)%, (59.3 ± 2.8)%, (72.7 ± 4.8)%, (78.2 ± 5.9)%, (63.3 ± 2.9)% and (43.2 ± 2.6)%, respectively; while those were (18.5 ± 1.2)%, (22.3 ± 1.7)%, (3.6 ± 2.5)%, (24.8 ± 2.3)%, (22.3 ± 1.3)% and (23.8 ± 1.8)% after blocked by PLC, respectively. There were no changes of the microtubule observed in DC2.4 cells at the same time points. The rates of the F-actin rearrangements before blocked by PLC were higher than those after PLC blockade at 4, 6, 8 and 10 h (P <0.05). The expressions of PLC in cytomembrane in DC2.4 cells increased after 2 h and reached its highest level at 8 h. The PLC inhibitor U73122 inhibited the expressions of PCL in cytomembrane of DC2.4 cells, but not in cytoplasm.</p><p><b>CONCLUSION</b>Mycobacterium tuberculosis can provoke to F-actin rearrangements through PLC molecule, which would further lead to Mycobacterium tuberculosis invasion of DC2.4 cells.</p>

Cytosolic Ca(2+) as a multifunctional modulator is required for spermiogenesis in Ascaris suum

The dynamic polar polymers actin filaments and microtubules are usually employed to provide the structural basis for establishing cell polarity in most eukaryotic cells. Radially round and immotile spermatids from nematodes contain almost no actin or tubulin, but still have the ability to break symmetry to extend a pseudopod and initiate the acquisition of motility powered by the dynamics of cytoskeleton composed of major sperm protein (MSP) during spermiogenesis (sperm activation). However, the signal transduction mechanism of nematode sperm activation and motility acquisition remains poorly understood. Here we show that Ca(2+) oscillations induced by the Ca(2+) release from intracellular Ca(2+) store through inositol (1,4,5)-trisphosphate receptor are required for Ascaris suum sperm activation. The chelation of cytosolic Ca(2+) suppresses the generation of a functional pseudopod, and this suppression can be relieved by introducing exogenous Ca(2+) into sperm cells. Ca(2+) promotes MSP-based sperm motility by increasing mitochondrial membrane potential and thus the energy supply required for MSP cytoskeleton assembly. On the other hand, Ca(2+) promotes MSP disassembly by activating Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase calcineurin. In addition, Ca(2+)/camodulin activity is required for the fusion of sperm-specifi c membranous organelle with the plasma membrane, a regulated exocytosis required for sperm motility. Thus, Ca(2+) plays multifunctional roles during sperm activation in Ascaris suum.