Humans ; Colorectal Neoplasms/genetics* ; Esophageal Neoplasms ; Genetic Predisposition to Disease ; Genotype ; Phosphoinositide Phospholipase C/genetics* ; Polymorphism, Single Nucleotide

Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; MicroRNAs/genetics* ; Phospholipase C delta ; Prostatic Neoplasms/genetics*

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.
Acetylcholine ; Animals ; Atropine ; Blood Glucose ; Calcium Channels ; Humans ; Injections, Intraperitoneal ; Muscle, Smooth* ; Papaverine ; Prazosin ; Protein Kinase C ; Rats* ; Receptor, Adenosine A1 ; Receptors, Muscarinic ; Streptozocin ; Type C Phospholipases ; Urinary Bladder* ; Verapamil

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.
Calcium ; Cytoplasm ; Endoplasmic Reticulum ; GTP-Binding Proteins ; HEK293 Cells ; Inositol ; Phosphatidylinositol 4,5-Diphosphate ; Phospholipases ; Phosphotransferases ; Protein Kinase C ; Transient Receptor Potential Channels ; Type C Phospholipases

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.
Animals ; Calcium ; Cats* ; Egtazic Acid ; Fura-2 ; Methods ; Microscopy, Fluorescence ; Muscle Contraction ; Muscle, Smooth* ; Myocytes, Smooth Muscle* ; Nimodipine ; Pertussis Toxin ; Phospholipases ; Sarcoplasmic Reticulum ; Thapsigargin ; Type C Phospholipases

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.
Blotting, Western ; Cell Line ; Down-Regulation ; Epithelial Cells ; Esophageal Neoplasms* ; NF-kappa B ; Protein Kinase C-alpha ; RNA, Small Interfering ; Type C Phospholipases

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.
Animals ; Atropine ; Diabetes Mellitus ; Muscle, Smooth ; Phosphotransferases ; Prazosin ; Rats* ; Rats, Sprague-Dawley ; Streptozocin ; Type C Phospholipases ; Urinary Bladder* ; Verapamil

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.
Apoptosis ; Cell Proliferation ; Corneal Injuries ; Epithelium, Corneal ; Humans ; In Vitro Techniques ; Inositol 1,4,5-Trisphosphate ; Mortuary Practice ; Panax ; Rabbits ; Receptors, Lysophosphatidic Acid ; Signal Transduction ; Therapeutic Uses ; Type C Phospholipases ; Wound Healing* ; Wounds and Injuries*

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.
Animals ; Calcium ; Dermatitis, Atopic ; Humans ; Ion Channels ; Ketanserin ; Mice ; Serotonin* ; Skin ; Type C Phospholipases

Angiotensin II (Ang II) is metabolized from N-terminal by aminopeptidases and from C-terminal by Ang converting enzyme (ACE) to generate several truncated angiotensin peptides (Angs). The truncated Angs have different biological effects but it remains unknown whether Ang-(4-8) is an active peptide. The present study was to investigate the effects of Ang-(4-8) on hemodynamics and atrial natriuretic peptide (ANP) secretion using isolated beating rat atria. Atrial stretch caused increases in atrial contractility by 60% and in ANP secretion by 70%. Ang-(4-8) (0.01, 0.1, and 1 µM) suppressed high stretch-induced ANP secretion in a dose-dependent manner. Ang-(4-8) (0.1 µM)-induced suppression of ANP secretion was attenuated by the pretreatment with an antagonist of Ang type 1 receptor (AT₁R) but not by an antagonist of AT₂R or AT₄R. Ang-(4-8)-induced suppression of ANP secretion was attenuated by the pretreatment with inhibitor of phospholipase (PLC), inositol triphosphate (IP₃) receptor, or nonspecific protein kinase C (PKC). The potency of Ang-(4-8) to inhibit ANP secretion was similar to Ang II. However, Ang-(4-8) 10 µM caused an increased mean arterial pressure which was similar to that by 1 nM Ang II. Therefore, we suggest that Ang-(4-8) suppresses high stretch-induced ANP secretion through the AT₁R and PLC/IP₃/PKC pathway. Ang-(4-8) is a biologically active peptide which functions as an inhibition mechanism of ANP secretion and an increment of blood pressure.
Aminopeptidases ; Angiotensin II* ; Angiotensins* ; Animals ; Arterial Pressure ; Atrial Natriuretic Factor* ; Blood Pressure* ; Heart ; Hemodynamics ; Inositol ; Peptides ; Phospholipases ; Protein Kinase C ; Rats* ; Receptor, Angiotensin, Type 1 ; Signal Transduction