Cellular Mechanism Underlying the Facilitation of Contractile Response Induced by Tumor Necrosis Factor-α in Mouse Tracheal Smooth Muscle.

The proinflammatory cytokine tumor necrosis factor-α (TNF-α) augments intracellular Ca2+ signaling and contractile responses of airway smooth muscles, leading to airway hyperresponsiveness. However, the underlying mechanism has not been fully elucidated. This study aimed to investigate the cellular mechanism of the potentiated contraction of mouse tracheal smooth muscle induced by TNF-α. The results showed that TNF-α triggered facilitation of mouse tracheal smooth muscle contraction in an epithelium-independent manner. The TNF-α-induced hypercontractility could be suppressed by the protein kinase C inhibitor GF109203X, the tyrosine kinase inhibitor genistein, the Src inhibitor PP2, or the L-type voltage-dependent Ca2+ channel blocker nifedipine. Following TNF-α incubation, the α1C L-type Ca2+ channel (CaV1.2) was up-regulated in cultured primary mouse tracheal smooth muscle cells. Pronounced phosphotyrosine levels were observed in mouse tracheas. In conclusion, this study shows that TNF-α enhanced airway smooth muscle contraction via protein kinase C-Src-CaV1.2 pathways, which provides novel insights into the pathologic role of proinflammatory cytokines in mediating airway hyperresponsiveness.

Rapid shifts in thermal reaction norms and tolerance of brooded coral larvae following parental heat acclimation.

Thermal priming of reef corals can enhance their heat tolerance; however, the legacy effects of heat stress during parental brooding on larval resilience remain understudied. This study investigated whether preconditioning adult coral Pocillopora damicornis to high temperatures (29°C and 32°C) could better prepare their larvae for heat stress. Results showed that heat-acclimated adults brooded larvae with reduced symbiont density and shifted thermal performance curves. Reciprocal transplant experiments demonstrated higher bleaching resistance and better photosynthetic and autotrophic performance in heat-exposed larvae from acclimated adults compared to unacclimated adults. RNA-seq revealed strong cellular stress responses in larvae from heat-acclimated adults that could have been effective in rescuing host cells from stress, as evidenced by the widespread upregulation of genes involved in cell cycle and mitosis. For symbionts, a molecular coordination between light harvesting, photoprotection and carbon fixation was detected in larvae from heat-acclimated adults, which may help optimize photosynthetic activity and yield under high temperature. Furthermore, heat acclimation led to opposing regulations of symbiont catabolic and anabolic pathways and favoured nutrient translocation to the host and thus a functional symbiosis. Notwithstanding, the improved heat tolerance was paralleled by reduced light-enhanced dark respiration, indicating metabolic depression for energy saving. Our findings suggest that adult heat acclimation can rapidly shift thermal tolerance of brooded coral larvae and provide integrated physiological and molecular evidence for this adaptive plasticity, which could increase climate resilience. However, the metabolic depression may be maladaptive for long-term organismal performance, highlighting the importance of curbing carbon emissions to better protect corals.

Cellular mechanism underlying the facilitation of contractile response induced by IL-25 in mouse tracheal smooth muscle.

Asthma is a common heterogeneous respiratory disease characterized by airway inflammation and airway hyperresponsiveness (AHR) which is associated with abnormality in smooth muscle contractility. The epithelial cell-derived cytokine IL-25 is implicated in type 2 immune pathology including asthma, whereas the underlying mechanisms have not been fully elucidated. This study aims to investigate the effects of IL-25 on mouse tracheal smooth muscle contractility and elucidate the cellular mechanisms. Incubation with IL-25 augmented the contraction of mouse tracheal smooth muscles, which could be suppressed by the L-type voltage-dependent Ca2+ channel (L-VDCC) blocker nifedipine. Furthermore, IL-25 enhanced the cytosolic Ca2+ signals and triggered the upregulation of α1C L-VDCC (CaV1.2) in primary cultured mouse tracheal smooth muscle cells. Knocking down IL-17RA/IL-17RB receptors or inhibiting the transforming growth factor-ß-activated kinase 1 (TAK1)-tumor progression locus 2 (TPL2)-MAPK kinase 1/2 (MEK1/2)-ERK1/2-activating protein-1 (AP-1) signaling pathways suppressed the IL-25-elicited upregulation of CaV1.2 and hyperreactivity in tracheal smooth muscles. Moreover, inhibition of TPL2, ERK1/2 or L-VDCC alleviated the AHR symptom induced by IL-25 in a murine model. This study revealed that IL-25 potentiated the contraction of tracheal smooth muscle and evoked AHR via activation of TPL2-ERK1/2-CaV1.2 signaling, providing novel targets for the treatment of asthma with a high-IL-25 phenotype.

G protein-coupled estrogen receptor promotes acrosome reaction via regulation of Ca2+ signaling in mouse sperm†.

G protein-coupled estrogen receptor (GPER), a seven-transmembrane G protein-coupled receptor, mediates the rapid pre-genomic signaling actions of estrogen and derivatives thereof. The expression of GPER is extensive in mammal male reproductive system. However, the functional role of GPER in mouse sperm has not yet been well recognized. This study revealed that GPER was expressed at the acrosome and the mid-flagellum of the mouse sperm. The endogenous GPER ligand 17ß-estradiol and the selective GPER agonist G1 increased intracellular Ca2+ concentration ([Ca2+]i) in mouse sperm, which could be abolished by G15, an antagonist of GPER. In addition, the G1-stimulated Ca2+ response was attenuated by interference with the phospholipase C (PLC) signaling pathways or by blocking the cation channel of sperm (CatSper). Chlortetracycline staining assay showed that the activation of GPER increased the incidence of acrosome-reacted sperm. Conclusively, GPER was located at the acrosome and mid-flagellum of the mouse sperm. Activation of GPER triggered the elevation of [Ca2+]i through PLC-dependent Ca2+ mobilization and CatSper-mediated Ca2+ influx, which promoted the acrosome reaction of mouse sperm.

Activation of TRPV4 stimulates transepithelial K+ secretion in rat epididymal epithelium.

The maturation of sperms is dependent on the coordinated interactions between sperm and the unique epididymal luminal milieu, which is characterized by high K+ content. This study investigated the involvement of transient receptor potential vanilloid 4 (TRPV4) in the K+ secretion of epididymal epithelium. The expression level and cellular localization of TRPV4 and Ca2+-activated K+ channels (KCa) were analyzed via RT-PCR, real-time quantitative PCR, western blot and immunofluorescence. The functional role of TRPV4 was investigated using short-circuit current (ISC) and intracellular Ca2+ imaging techniques. We found a predominant expression of TRPV4 in the corpus and cauda epididymal epithelium. Activation of TRPV4 with a selective agonist, GSK1016790A, stimulated a transient decrease in the ISC of the epididymal epithelium. The ISC response was abolished by either the TRPV4 antagonists, HC067047 and RN-1734, or the removal of basolateral K+. Simultaneously, the application of GSK1016790A triggered Ca2+ influx in epididymal epithelial cells. Our data also indicated that the big conductance KCa (BK), small conductance KCa (SK) and intermediate conductance KCa (IK) were all expressed in rat epididymis. Pharmacological studies revealed that BK, but not SK and IK, mediated TRPV4-elicited transepithelial K+ secretion. Finally, we demonstrated that TRPV4 and BK were localized in the epididymal epithelium, which showed an increased expression level from caput to cauda regions of rat epididymis. This study implicates that TRPV4 plays an important role in the formation of high K+ concentration in epididymal intraluminal fluid via promoting transepithelial K+ secretion mediated by BK.

Local glutamate-mediated dendritic plateau potentials change the state of the cortical pyramidal neuron.

Dendritic spikes in thin dendritic branches (basal and oblique dendrites) are traditionally inferred from spikelets measured in the cell body. Here, we used laser-spot voltage-sensitive dye imaging in cortical pyramidal neurons (rat brain slices) to investigate the voltage waveforms of dendritic potentials occurring in response to spatially restricted glutamatergic inputs. Local dendritic potentials lasted 200-500 ms and propagated to the cell body, where they caused sustained 10- to 20-mV depolarizations. Plateau potentials propagating from dendrite to soma and action potentials propagating from soma to dendrite created complex voltage waveforms in the middle of the thin basal dendrite, comprised of local sodium spikelets, local plateau potentials, and backpropagating action potentials, superimposed on each other. Our model replicated these voltage waveforms across a gradient of glutamatergic stimulation intensities. The model then predicted that somatic input resistance (Rin) and membrane time constant (tau) may be reduced during dendritic plateau potential. We then tested these model predictions in real neurons and found that the model correctly predicted the direction of Rin and tau change but not the magnitude. In summary, dendritic plateau potentials occurring in basal and oblique branches put pyramidal neurons into an activated neuronal state ("prepared state"), characterized by depolarized membrane potential and smaller but faster membrane responses. The prepared state provides a time window of 200-500 ms, during which cortical neurons are particularly excitable and capable of following afferent inputs. At the network level, this predicts that sets of cells with simultaneous plateaus would provide cellular substrate for the formation of functional neuronal ensembles.NEW & NOTEWORTHY In cortical pyramidal neurons, we recorded glutamate-mediated dendritic plateau potentials with voltage imaging and created a computer model that recreated experimental measures from dendrite and cell body. Our model made new predictions, which were then tested in experiments. Plateau potentials profoundly change neuronal state: a plateau potential triggered in one basal dendrite depolarizes the soma and shortens membrane time constant, making the cell more susceptible to firing triggered by other afferent inputs.

Cellular mechanism underlying oxytocin-stimulated Cl- secretion in rat cauda epididymal epithelium.

The neurohypophyseal hormone oxytocin (OT) plays critical roles in lactation and parturition, while its function in male reproduction system is largely unknown. This study aims to investigate the effect of OT on regulating transepithelial ion transport in rat cauda epididymal epithelium. With the use of RT-PCR, Western blot, and immunohistochemical analysis, we found that OT receptor (OTR) was expressed and localized at the basal membrane of rat cauda epididymal epithelium. The short-circuit current (Isc) measurement showed that basolateral application of OT to the primary cultured rat cauda epididymal epithelial cells elicited an increase in Isc, which was abrogated by pretreating the epithelial cells with CFTRinh-172, a blocker of cystic fibrosis transmembrane conductance regulator (CFTR). Pretreatment with the prostaglandin H synthase inhibitors indomethacin and piroxicam, or the nonselective antagonists of prostaglandin E2 (PGE2) receptor EP2 or EP4, AH-6809, and AH-23848, significantly attenuated OT-stimulated Isc response. Furthermore, the generation of PGE2 was measured using enzyme-linked immunosorbent assay, demonstrating that OT induced a substantial increase in PGE2 release from primary cultured rat cauda epididymal epithelial cells. In conclusion, activation of OTR by OT triggered PGE2 release, resulting in CFTR-dependent Cl- secretion through paracrine/autocrine pathways in rat cauda epididymal epithelium.

Cellular mechanisms underlying carbon monoxide stimulated anion secretion in rat epididymal epithelium.

Epididymal epithelium possesses active ion transport properties conducive to the maintenance of appropriate epididymal intraluminal microenvironment. The endogenous gasotransmitter carbon monoxide (CO) regulates numerous cellular processes including water and electrolyte transport in various epithelia. However, the functional role of CO in epididymal epithelium is still elusive. This study aims to explore the potential regulatory effect of CO on transepithelial ion transport in rat epididymis. Using qPCR technique, we verified that endogenous CO synthase heme oxygenase 1 was expressed in rat caput, corpus, and cauda epididymis. In addition, endogenous CO was detected in rat cauda epididymis. Ussing chamber experiments showed that CORM-2, a CO donor, induced an increase of the short-circuit current (ISC) in a concentration-dependent manner in rat cauda epididymal epithelium. The ISC response could be abrogated by removing the ambient Cl- or HCO3-. Interfering with the cAMP signaling pathway or blocking cystic fibrosis transmembrane regulator (CFTR) partially suppressed the CO-stimulated ISC response. Moreover, the CO-evoked ISC response was significantly attenuated by blocking Ca2+-activated Cl- channel (CaCC) or chelating intracellular Ca2+. Elevation of intracellular Ca2+ level was also observed after CO stimulation in rat cauda epididymal epithelial cells. Collectively, this study demonstrated that CO stimulated anion secretion via activation of CFTR and CaCC in rat cauda epididymal epithelium, which might contribute to the formation of the appropriate microenvironment essential for sperm storage.

Mechanisms underlying the regulation of intracellular and luminal pH in vaginal epithelium.

The vagina provides a characteristic low-Na+ and low-pH fluid microenvironment that is considered generally protective. Previous studies have shown that various types of epithelial cells harbor the capacity of intracellular pH (pHi) regulation. However, it remains elusive whether vaginal epithelium could actively regulate pHi by transporting acid-base ions. In this study, we verified that after transient exposure to NH4 Cl, the pHi values could rapidly recover from acidification via Na+ -H+ exchanger (NHE), Na+ -HCO3 - cotransporter (NBC), and carbonic anhydrase in human vaginal epithelial cell line VK2/E6E7. Positive expression of the main acid-base transporters including NHE1-2, NBCe1-2, and NBCn1 mRNA was also detected in VK2/E6E7 cells. Moreover, the in vivo study further showed that interfering with the function of V-type H+ -ATPase, NHE or NBC expressed in vagina impaired vaginal luminal pH homeostasis in rats. Taken together, our study reveals the property of pH regulation in vaginal epithelial cells, which might provide novel insights into the potential role of vaginal epithelium in the formation of the vaginal acidic microenvironment.

The gasotransmitter hydrogen sulfide inhibits transepithelial anion secretion of pregnant mouse endometrial epithelium.

Endometrial epithelium exhibits a robust ion transport activity required for dynamical regulation of uterine fluid environment and thus embryo implantation. However, there still lacks a thorough understanding of the ion transport processes and regulatory mechanism in peri-implantation endometrial epithelium. As a gaseous signaling molecule or gasotransmitter, hydrogen sulfide (H2S) regulates a myriad of cellular and physiological processes in various tissues, including the modulation of ion transport proteins in epithelium. This study aimed to investigate the effects of H2S on ion transport across mouse endometrial epithelium and its possible role in embryo implantation. The existence of endogenous H2S in pregnant mouse uterus was tested by the detection of two key H2S-generating enzymes and measurement of H2S production rate in tissue homogenates. Transepithelial ion transport processes were electrophysiologically assessed in Ussing chambers on early pregnant mouse endometrial epithelial layers, demonstrating that H2S suppressed the anion secretion by blocking cystic fibrosis transmembrane conductance regulator (CFTR). H2S increased intracellular Cl- concentration ([Cl-]i) in mouse endometrial epithelial cells, which was abolished by pretreatment with the CFTR selective inhibitor CFTRinh-172. The cAMP level in mouse endometrial epithelial cells was not affected by H2S, indicating that H2S blocked CFTR in a cAMP-independent way. In vivo study showed that interference with H2S synthesis impaired embryo implantation. In conclusion, our study demonstrated that H2S inhibits the transepithelial anion secretion of early pregnant mouse endometrial epithelium via blockade of CFTR, contributing to the preparation for embryo implantation.

[Expression of the NMDA receptor in the rat model of orchialgia].

OBJECTIVE: To explore the expression of the N-methyl-D-aspartate (NMDA) receptor in the rat model of orchialgia and its possible mechanisms. METHODS: According to Yoshioka's method, the male rats in the control group were injected with 0.2 ml saline, and those in the experimental group with 0.2 ml 2% acetic acid solution. Then we tested the behavioral responses of the rats and determined the expressions of the subunits NR1 and NR2B of the NMDA receptor in the dorsal root ganglion and spinal dorsal horn by Western blot, RT-qPCR and immunofluorescence staining. RESULTS: The withdrawal latency was decreased in the model rats, reaching the lowest value at 4 hours after modeling, significantly lower than in the controls (ï¼»4.15 ± 0.84ï¼½ vs ï¼»12.32 ± 1.05ï¼½, P < 0.05). Compared with the controls, the model rats showed remarkably increased mRNA and protein expressions of NR2B in the dorsal root ganglion (P < 0.05) but not in the spinal dorsal horn at 4 hours. However, no statistically significant difference was found in the expression of NR1 either in the dorsal root ganglion or in the spinal dorsal horn between the two groups (P > 0.05). CONCLUSIONS: The NMDA receptor plays an important role in pathogenesis of orchialgia in rats. In the early stage of pain, upregulating the expression of the subunit NR2B of the NMDA receptor can mediate peripheral hyperalgesia and consequently orchialgia.

[Oxidative stress induces damage to epididymal epithelial tight junction protein ZO-1 and impairs epididymal function in varicocele rats].

OBJECTIVE: To investigate oxidative stress-mediated damage to the epididymal epithelial tight junction protein ZO-1 and its impact on epididymal function in varicocele rats. METHODS: We randomly divided 45 male adolescent SD rats into three groups of equal number: sham operation (left renal vein exposed and isolated), experimental (left renal vein constricted and collaterals of the left spermatic vein fully ligated), and treatment (60-day intragastric administration of vitamin E at 150 mg/kg/d after modeling). At 60 days after modeling, we observed the histological changes in the left epididymis, detected the expressions of ZO-1 and other tight junction-related proteins by real-time quantitative PCR, immunohistochemistry, immunofluorescence staining and Western blotting, determined sperm motility, and measured the levels of superoxide dismutase (SOD), total antioxidant capacity (T-AOC), methylene dioxyamphetamine (MDA) and α-glucosidase (α-Glu) in the epididymal tissue of the rats. RESULTS: Compared with the rats of the sham operation group, those of the experimental group showed disorganized epithelial structure and decreased number of epithelial cells in the left epididymis, with some epithelial cells desquamated into the lumen. The expression of ZO-1 was significantly lower in the experimental than in the sham operation group (P < 0.05) but markedly upregulated after VE treatment (P < 0.05). In comparison with the sham operation group, the animals in the experimental group exhibited remarkably increased content of MDA in the epididymal tissue (ï¼»0.41 ± 0.05ï¼½ vs ï¼»1.21 ± 0.18ï¼½ nmol/mg prot, P < 0.05) but decreased levels of SOD (ï¼»814.65 ± 73.64ï¼½ vs ï¼»298.62 ± 67.84ï¼½ U/mg prot, P < 0.05), T-AOC (ï¼»0.84 ± 0.07ï¼½ vs ï¼»0.24 ± 0.04ï¼½ nmol/mg prot, P < 0.05) and α-Glu (ï¼»11.72 ± 2.72ï¼½ vs ï¼»5.82 ± 1.24ï¼½ U/mg prot, P < 0.05). VE treatment, however, remarkably reduced the content of MDA (ï¼»0.69 ± 0.12ï¼½ nmol/mg prot) and elevated the levels of SOD (ï¼»497.73 ± 48.03ï¼½ U/mg prot), T-AOC (ï¼»0.42 ± 0.06ï¼½ nmol/mg prot) and α-Glu (ï¼»9.11 ± 1.91ï¼½ U/mg prot) as compared with those in the experimental group (all P < 0.05). The percentage of progressively motile sperm was significantly lower in the experimental than in the sham operation group (ï¼»31.33 ± 6.32ï¼½% vs ï¼»71.21 ± 5.21ï¼½%, P < 0.05), but markedly increased after VE treatment (ï¼»60.68 ± 5.31ï¼½%, P < 0.05). CONCLUSIONS: Varicocele reduces the expression of the EETJ protein ZO-1 and impairs epididymal function via oxidative stress, while vitamin E can effectively upregulate the ZO-1 expression and improve epididymal function by decreasing oxidative stress in the epididymis of varicocele rats.

Cancer in the parasitic protozoans Trypanosoma brucei and Toxoplasma gondii.

Cancer is a general name for more than 100 malignant diseases. It is postulated that all cancers start from a single abnormal cell that grows out of control. Untreated cancers can cause serious consequences and deaths. Great progress has been made in cancer research that has significantly improved our knowledge and understanding of the nature and mechanisms of the disease, but the origins of cancer are far from being well understood due to the limitations of suitable model systems and to the complexities of the disease. In view of the fact that cancers are found in various species of vertebrates and other metazoa, here, we suggest that cancer also occurs in parasitic protozoans such as Trypanosoma brucei, a blood parasite, and Toxoplasma gondii, an obligate intracellular pathogen. Without treatment, these protozoan cancers may cause severe disease and death in mammals, including humans. The simpler genomes of these single-cell organisms, in combination with their complex life cycles and fascinating life cycle differentiation processes, may help us to better understand the origins of cancers and, in particular, leukemias.

Infection by Toxoplasma gondii, a severe parasite in neonates and AIDS patients, causes impaired anion secretion in airway epithelia.

The airway epithelia initiate and modulate the inflammatory responses to various pathogens. The cystic fibrosis transmembrane conductance regulator-mediated Cl(-) secretion system plays a key role in mucociliary clearance of inhaled pathogens. We have explored the effects of Toxoplasma gondii, an opportunistic intracellular protozoan parasite, on Cl(-) secretion of the mouse tracheal epithelia. In this study, ATP-induced Cl(-) secretion indicated the presence of a biphasic short-circuit current (Isc) response, which was mediated by a Ca(2+)-activated Cl(-) channel (CaCC) and the cystic fibrosis transmembrane conductance regulator. However, the ATP-evoked Cl(-) secretion in T. gondii-infected mouse tracheal epithelia and the elevation of [Ca(2+)]i in T. gondii-infected human airway epithelial cells were suppressed. Quantitative reverse transcription-PCR revealed that the mRNA expression level of the P2Y2 receptor (P2Y2-R) increased significantly in T. gondii-infected mouse tracheal cells. This revealed the influence that pathological changes in P2Y2-R had on the downstream signal, suggesting that P2Y2-R was involved in the mechanism underlying T. gondii infection in airways. These results link T. gondii infection as well as other pathogen infections to Cl(-) secretion, via P2Y2-R, which may provide new insights for the treatment of pneumonia caused by pathogens including T. gondii.

The stochastic nature of action potential backpropagation in apical tuft dendrites.

In cortical pyramidal neurons, backpropagating action potentials (bAPs) supply Ca2+ to synaptic contacts on dendrites. To determine whether the efficacy of AP backpropagation into apical tuft dendrites is stable over time, we performed dendritic Ca2+ and voltage imaging in rat brain slices. We found that the amplitude of bAP-Ca2+ in apical tuft branches was unstable, given that it varied from trial to trial (termed "bAP-Ca2+ flickering"). Small perturbations in dendritic physiology, such as spontaneous synaptic inputs, channel inactivation, or temperature-induced changes in channel kinetics, can cause bAP flickering. In the tuft branches, the density of Na+ and K+ channels was sufficient to support local initiation of fast spikelets by glutamate iontophoresis. We quantified the time delay between the somatic AP burst and the peak of dendritic Ca2+ transient in the apical tuft, because this delay is important for induction of spike-timing dependent plasticity. Depending on the frequency of the somatic AP triplets, Ca2+ signals peaked in the apical tuft 20-50 ms after the 1st AP in the soma. Interestingly, at low frequency (<20 Hz), the Ca2+ peaked sooner than at high frequency, because only the 1st AP invaded tuft. Activation of dendritic voltage-gated Ca2+ channels is sensitive to the duration of the dendritic voltage transient. In apical tuft branches, small changes in the duration of bAP voltage waveforms cause disproportionately large increases in dendritic Ca2+ influx (bAP-Ca2+ flickering). The stochastic nature of bAP-Ca2+ adds a new perspective on the mechanisms by which pyramidal neurons combine inputs arriving at different cortical layers.NEW & NOTEWORTHY The bAP-Ca2+ signal amplitudes in some apical tuft branches randomly vary from moment to moment. In repetitive measurements, successful AP invasions are followed by complete failures. Passive spread of voltage from the apical trunk into the tuft occasionally reaches the threshold for local Na+ spike, resulting in stronger Ca2+ influx. During a burst of three somatic APs, the peak of dendritic Ca2+ in the apical tuft occurs with a delay of 20-50 ms depending on AP frequency.

Naringenin Regulates CFTR Activation and Expression in Airway Epithelial Cells.

BACKGROUND/AIMS: Sputum symptoms are commonly seen in the elderly. This study aimed to identify an efficacious expectorant treatment stratagem through evaluating the secretion-promoting activation and cystic fibrosis transmembrane conductance regulator (CFTR) expression of the bioactive herbal monomer naringenin. METHODS: Vectorial Cl- transport was determined by measuring short-circuit current (ISC) in rat airway epithelium. cAMP content was measured by ELISA in primary cultured epithelial cells and Calu-3 cells. CFTR expression in Calu-3 cells was determined by qPCR. RESULTS: Addition of naringenin to the basolateral side of the rat airway led to a concentration-dependent sustained increase in ISC. The current was suppressed when exposed to Cl--free solution or by bumetanide, BaCl2, and DPC but not by DIDS and IBMX. Forskolin-induced ISC increase and CFTRinh-172/MDL-12330A-induced ISC inhibition were not altered by naringenin. Intracellular cAMP content was significantly increased by naringenin. With lipopolysaccharide stimulation, CFTR expression was significantly reduced, and naringenin dose-dependently enhanced CFTR mRNA expression. CONCLUSION: These results demonstrate that naringenin has the ability to stimulate Cl- secretion, which is mediated by CFTR through a signaling pathway by increasing cAMP content. Moreover, naringenin can increase CFTR expression when organism CFTR expression is seriously hampered. Our data suggest a potentially effective treatment strategy for sputum.

Role of GPR30 in estrogen-induced prostate epithelial apoptosis and benign prostatic hyperplasia.

Several studies have implicated estrogen and the estrogen receptor (ER) in the pathogenesis of benign prostatic hyperplasia (BPH); however, the mechanism underlying this effect remains elusive. In the present study, we demonstrated that estrogen (17ß-estradiol, or E2)-induced activation of the G protein-coupled receptor 30 (GPR30) triggered Ca2+ release from the endoplasmic reticulum, increased the mitochondrial Ca2+ concentration, and thus induced prostate epithelial cell (PEC) apoptosis. Both E2 and the GPR30-specific agonist G1 induced a transient intracellular Ca2+ release in PECs via the phospholipase C (PLC)-inositol 1, 4, 5-triphosphate (IP3) pathway, and this was abolished by treatment with the GPR30 antagonist G15. The release of cytochrome c and activation of caspase-3 in response to GPR30 activation were observed. Data generated from the analysis of animal models and human clinical samples indicate that treatment with the GPR30 agonist relieves testosterone propionate (TP)-induced prostatic epithelial hyperplasia, and that the abundance of GPR30 is negatively associated with prostate volume. On the basis of these results, we propose a novel regulatory mechanism whereby estrogen induces the apoptosis of PECs via GPR30 activation. Inhibition of this activation is predicted to lead to abnormal PEC accumulation, and to thereby contribute to BPH pathogenesis.

Functional expression of G protein-coupled receptor 30 in immature rat epididymal epithelium.

The aim of this study is to investigate the functional role of G protein-coupled receptor 30 (GPR30) in the epididymis. We found that GPR30 is expressed in the epithelium of the immature rat epididymis and is involved in chloride secretion into the caudal epididymis lumen. The short-circuit current (Isc) experiments showed that in primary cultured caudal epididymis epithelium, activation of GPR30 by its specific agonist G1 induced a mono-phasic current increase, and G15, the specific antagonist of GPR30, could completely inhibit the current induced by G1. The G1-induced Isc was largely blocked by application of the non-specific chloride channel inhibitor diphenylamine-dicarboxylic acid (DPC), or by the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh-172 , suggesting that the current was mainly mediated through CFTR. In addition, after stimulating GPR30 by G1, the intracellular concentration of cAMP in the epithelium was significantly increased, indicating that the cAMP signal pathway is involved and could be responsible for the CFTR activation. Finally, to further investigate the function of GPR30 in vivo, G15 was administrated into rats subcutaneously. The osmotic pressure of the micro perfusion solution from epididymis was measured and the sperms were collected. Results showed that there was an osmotic pressure increase of the perfusion solution from G15 treated rats. When the GPR30 was inhibited by G15 endogenously, the motility of sperms decreased. Our data demonstrated that GPR30 is involved in the formation of caudal epididymis fluid micro-environment thus affecting sperm motility.

Relaxant Effect of Sodium Tanshinone IIA Sulphonate on Mouse Tracheal Smooth Muscle.

Sodium tanshinone IIA sulphonate, a water-soluble derivative of tanshinone IIA, has been proven to possess versatile biological properties, but its pharmacological effect on tracheal smooth muscle remains elusive. This paper presents a study on the relaxant effect and underlying mechanisms of sodium tanshinone IIA sulphonate on mouse tracheal smooth muscle. The relaxant effect of sodium tanshinone IIA sulphonate was evaluated in mouse tracheal rings using a mechanical recording system. Intracellular Ca2+ concentration was measured in primary cultured tracheal smooth muscle cells using confocal imaging system. The results showed that sodium tanshinone IIA sulphonate induced dose-dependent relaxation of mouse tracheal rings in a ß-adrenoceptor- and epithelium-independent manner. Pretreatment with the ATP-sensitive K+ channel blocker glibenclamide partly attenuated the relaxation response. Administration of sodium tanshinone IIA sulphonate notably inhibited the extracellular Ca2+-induced contraction. High KCl or carbachol-evoked elevation in the intracellular Ca2+ concentration was also abrogated by sodium tanshinone IIA sulphonate in tracheal smooth muscle cells. In conclusion, the tracheal relaxant effect of sodium tanshinone IIA sulphonate was independent of ß-adrenoceptor and airway epithelium, mediated primarily by inhibition of extracellular Ca2+ influx via L-type voltage-dependent Ca2+ channels and partially by activation of the ATP-sensitive K+ channel. These results indicate the potential therapeutic value of sodium tanshinone IIA sulphonate for asthma treatment.

Connexin hemichannels contribute to spontaneous electrical activity in the human fetal cortex.

Before the human cortex is able to process sensory information, young postmitotic neurons must maintain occasional bursts of action-potential firing to attract and keep synaptic contacts, to drive gene expression, and to transition to mature membrane properties. Before birth, human subplate (SP) neurons are spontaneously active, displaying bursts of electrical activity (plateau depolarizations with action potentials). Using whole-cell recordings in acute cortical slices, we investigated the source of this early activity. The spontaneous depolarizations in human SP neurons at midgestation (17-23 gestational weeks) were not completely eliminated by tetrodotoxin--a drug that blocks action potential firing and network activity--or by antagonists of glutamatergic, GABAergic, or glycinergic synaptic transmission. We then turned our focus away from standard chemical synapses to connexin-based gap junctions and hemichannels. PCR and immunohistochemical analysis identified the presence of connexins (Cx26/Cx32/Cx36) in the human fetal cortex. However, the connexin-positive cells were not found in clusters but, rather, were dispersed in the SP zone. Also, gap junction-permeable dyes did not diffuse to neighboring cells, suggesting that SP neurons were not strongly coupled to other cells at this age. Application of the gap junction and hemichannel inhibitors octanol, flufenamic acid, and carbenoxolone significantly blocked spontaneous activity. The putative hemichannel antagonist lanthanum alone was a potent inhibitor of the spontaneous activity. Together, these data suggest that connexin hemichannels contribute to spontaneous depolarizations in the human fetal cortex during the second trimester of gestation.