High burden of human papillomavirus infection among men in Guangzhou, South China: Implications for HPV vaccination strategies.

The epidemiological and clinical aspects of Human Papillomavirus (HPV) infection in women have been extensively studied. However, there is a lack of information regarding HPV characteristics in males. In this study, we conducted a retrospective and observational study of 3737 consecutive male individuals attending outpatient clinics of Guangdong Women and Children Hospital from 2012 to 2023 in Guangzhou, South China, to determine the age- and genotype-specific prevalence of HPV in men. The results showed the overall prevalence of HPV among men was 42.15% (1575/3737), with variations ranging from 29.55% to 81.31% across distinct diagnostic populations. Low-risk HPV6 (15.47%), HPV11 (8.94%), and high-risk HPV52 (5.51%) were the most common types. The annual HPV prevalence decreased significantly (Z = -3.882, p < .001), ranging from 31.44% to 52.90%. 28.77% (1075/3737) of men manifested infection with a singular HPV type, predominantly identified as a low-risk type. The age-specific distribution of HPV infections revealed distinctive peaks in the < 25 y age group (47.60%, 208/437) and the 40-44 y age group (44.51%, 154/346). Notably, the positive rate of Chlamydia trachomatis was significantly higher among HPV-positive individuals in comparison to HPV-negatives (16.14% vs. 11.25%, p < .05). Our findings reveal a substantial prevalence of HPV infection among outpatient men in Guangzhou, South China. It is recommended to consider the inclusion of HPV vaccination for adolescent males in national immunization schedules, once an adequate supply of vaccines is accessible.

One-Step Synthesis of Hydrogel Adhesive with Acid-Responsive Tannin Release for Diabetic Oral Mucosa Defects Healing.

The complex preparation, weak wet tissue adhesion, and limited biological activity of traditional oral wound dressings usually impede their efficient treatment and healing for diabetic oral mucosal defects. To overcome these problems, a novel hydrogel adhesive (named CFT hydrogel) is rapidly constructed using a one-step method based on dual-dynamic covalent cross-linking. Compared with the commercial oral patches, the CFT hydrogel shows superior in vivo (rat tongue) wet tissue adhesion performance. Additionally, the CFT hydrogel exhibits unique acid-responsive properties, thereby facilitating the release of bioactive molecule tannic acid in the acidic diabetic wound microenvironment. And a series of in vitro experiments substantiate the favorable biocompatibility and bioactivity properties (including antibacterial, antioxidative, anti-inflammatory, and angiogenetic effects) exhibited by CFT hydrogel. Moreover, in vivo experiments conducted on a diabetic rat model with oral mucosal defects demonstrate that the CFT hydrogel exhibits significant efficacy in protecting against mucosal wounds, alleviating inflammatory reactions, thereby facilitating the wound-healing process. Taken together, this study provides a promising and comprehensive therapeutic option with great potential for the clinical management of oral mucosa defects in diabetic patients.

Role of Pseudomonas fluorescens FSYZ01 on the corrosion behavior of Q235B carbon steel in oilfield produced water.

The corrosion behavior of Q235B carbon steel is investigated in water, LB medium, and oilfield produced water adding Pseudomonas fluorescens FSYZ01. After immersion at 30 °C for 13 days, the weight loss of carbon steel with this strain decreased by 32.23%, 54.07%, and 78.34%, respectively. X-ray diffraction (XRD) results show that P. fluorescens FSYZ01 inhibited conversion of iron oxides by hindering oxygen from approaching metal surface. Fourier transform infrared (FT-IR) and X-ray photoelectron spectrometer (XPS) results show that specific functional groups and bonds reacted with Fe(II/III) to form a dense and stable chelate-oxide protective layer, thereby inhibiting corrosion. Pyrolysis gas chromatography-mass spectrometer (Py-GCMS) results demonstrate the bacteria degraded C12 to C20 alkanes in oil. The inhibitory mechanism of crude oil-degrading bacteria P. fluorescens FSYZ01 on the carbon steel corrosion was proposed, so as to slow corrosion of oilfield produced water system pipeline and prolong its service life, helping to comprehend the microbial corrosion in the actual environment.

Fabrication and characterization of PVA/CS-PCL/gel multi-scale electrospun scaffold: simulating extracellular matrix for enhanced cellular infiltration and proliferation.

A new bi-component poly(vinylalcohol)(PVA)/chitosan(CS)-poly(e-caprolactone)(PCL)/gelatin(Gel) multiscale electrospun scaffold was developed and analyzed in comparison with several other single scale systems. To mimic the native extracellular matrix in composition and structure and promote the migration of cells inside the scaffold, PVA/CS composite nanofibers (102 ± 52 nm) and PCL/Gelcomposite microfiber (2.5 ± 1.0 µm) were simultaneously electrospun from the two opposite syringes and mixed on a rotating mandrel to generate a bi-component multi-scale membrane. The bi-component membrane was crosslinked by glutaraldehyde vapor to maintain its fiber morphology in the wet stage. Morphology, shrinkage and spectroscopic of the electrospun membranes were characterized. To test the newly developed multiscale membrane, we seeded mesenchymal stem cells (MSCs) derived from rabbit onto five different fiber scaffolds (PVA, PVA/CS, PCL, PCL/Gel and PVA/CS-PCL/Gel) and compared cell adhesion and proliferation between different groups for 3 days using scanning electron microscopy, inverted microscope observations assay and MTT colorimetric. Cell culture results suggest that the incorporation of chitosan and gelatin could enhance cell adhesion and cell spreading in comparison to the performance of single component scaffolds of PVA and PCL. The multiscale PVA/CS-PCL/Gel membrane scaffolds provide a better environment to increase the growth, adhesion, and proliferation of cells. Scanning electron microscopy (SEM) observations showed that the cells were not only adhered well and proliferated on the surface of the scaffolds, but were also able to infiltrate inside the scaffold within 3 days of culture. MTT assay and inverted microscope observations also showed that the PVA/CS-PCL/Gel complex fibrous membrane exhibited better activity than other single component/scale systems scaffolds. Our results provide the underlying insights needed to guide the design of the native extracellular matrix.

Ginsenoside Rk1 induces apoptosis and downregulates the expression of PD-L1 by targeting the NF-κB pathway in lung adenocarcinoma.

Ginsenoside Rk1 is a substance derived from ginseng and exhibits various activities such as anti-diabetic, anti-inflammatory and anti-cancer effects; however, its anti-tumor effect and target signaling mechanism in lung adenocarcinoma are not well understood. Here, we show that Rk1, a natural drug product, can function as an antitumor modulator that induces apoptosis in lung adenocarcinoma cells by inhibiting NF-κB transcription and triggering cell cycle arrest. Mechanistically, Rk1 suppressed the proliferation and clonal formation of two lung adenocarcinoma cell lines (A549 and PC9) in vitro and caused G1 phase cell arrest. In the A549 xenograft model, Rk1 significantly inhibited tumor growth and had few toxic side effects on normal organs. Western blotting results showed that Rk1 increased the protein expression of Bax, cleaved caspase-3, -8, and -9, and PARP, decreased the expression of Bcl-2 and blocked the NF-κB signaling pathway. Furthermore, ginsenoside Rk1 also reduced the high expression of PD-L1 in lung adenocarcinoma cells by inhibiting NF-κB signaling. These data revealed a previously unreported antitumor mechanism of Rk1, providing new ideas and an experimental basis for further study of the mechanism of action of Rk1 in lung adenocarcinoma.

Folic acid-modified ginsenoside Rg5-loaded bovine serum albumin nanoparticles for targeted cancer therapy in vitro and in vivo.

BACKGROUND AND PURPOSE: Ginsenoside Rg5 (Rg5), a triterpene saponin, extracted from the natural herbal plant ginseng, is one of the most potent anticancer drugs against various carcinoma cells. However, the therapeutic potential of Rg5 is limited by its low solubility in water, poor bioavailability, and nontargeted delivery. Therefore, we prepared folic acid (FA)-modified bovine serum albumin (BSA) nanoparticles (FA-Rg5-BSA NPs) to improve the therapeutic efficacy and tumor targetability of Rg5. METHODS: Various aspects of the FA-Rg5-BSA NPs were characterized, including size, polydispersity, zeta potential, morphology, entrapment efficiency (EE), drug loading (DL), in vitro drug release, thermal stability, in vitro cytotoxicity, cell apoptosis, cellular uptake, in vivo antitumor effects and in vivo biodistribution imaging. RESULTS: The FA-Rg5-BSA NPs showed a particle size of 201.4 nm with a polydispersity index of 0.081, uniform spherical shape, and drug loading of 12.64±4.02%. The aqueous solution of FA-Rg5-BSA NPs had favorable stability for 8 weeks at 4°C. The FA-Rg5-BSA NPs dissolved under acidic conditions. Moreover, the Rg5-BSA NPs and FA-Rg5-BSA NPs had advanced anticancer activity compared with Rg5 in MCF-7 cells, while poor cytotoxicity was observed in L929 cells. The FA-Rg5-BSA NPs facilitated cellular uptake and induced apoptosis in MCF-7 cells. In addition, in an MCF-7 xenograft mouse model, the in vivo antitumor evaluation revealed that FA-Rg5-BSA NPs were more effective in inhibiting tumor growth than Rg5 and Rg5-BSA NPs. The in vivo real-time bioimaging study showed that the FA-Rg5-BSA NPs exhibited superior tumor accumulation ability. CONCLUSION: The results suggested that FA-Rg5-BSA NPs could serve as a promising system to improve the antitumor effect of Rg5.

Roles and Mechanisms of Human Cathelicidin LL-37 in Cancer.

LL-37, the C-terminal peptide of human cathelicidin antimicrobial peptide (CAMP, hCAP18), reportedly increases resistance to microbial invasion and exerts important physiological functions in chemotaxis, promotion of wound closure, and angiogenesis. Accumulating evidence indicates that LL-37 also plays a significant role in human cancer. LL-37 induces tumorigenic effects in cancers of the ovary, lung, breast, prostate, pancreas, as well as in malignant melanoma and skin squamous cell carcinoma. In contrast, LL-37 displays an anti-cancer effect in colon cancer, gastric cancer, hematologic malignancy and oral squamous cell carcinoma. Mechanistically, LL-37-induced activation of membrane receptors and subsequent signaling pathways lead to alteration of cellular functions. Different membrane receptors on various cancer cells appear to be responsible for the tissue-specific effects of LL-37. Meanwhile, the findings that vitamin D-dependent induction of cathelicidin in human macrophages activates the anti-cancer activity of tumor-associated macrophages (TAMs) and enhances antibody-dependent cellular cytotoxicity (ADCC) support critical roles of vitamin D-dependent induction of cathelicidin in cancer progression. This review describes novel advances involving the roles and mechanisms of human cathelicidin LL-37 in cancer.

DNA methylation directly downregulates human cathelicidin antimicrobial peptide gene (CAMP) promoter activity.

LL-37, the active product of human cathelicidin antimicrobial peptide (CAMP) has a broad spectrum of antibacterial activity. LL-37 also has important physiological functions in immune regulation, angiogenesis and in modulating apoptosis. The roles of LL-37 in oral squamous cell carcinoma (OSCC) are still not clear. The correlation between DNA methylation and human CAMP expression is also unknown. Here human CAMP/LL-37 expression was assessed by immunohistochemistry in normal and OSCC tissues. The results indicated that low expression of CAMP/LL-37 correlated with histological differentiation and lymph node metastasis and also promoted tumor progression. A cell-specific methylation pattern in the promoter region of human CAMP was detected. Treatment with 5-aza-2'-deoxycytidine, a DNA demethylation reagent can increase human CAMP expression in epithelial cancer cells. The reporter assay showed that unmethylated human CAMP promoter activity was significantly higher than methylated promoter activity. Taken together, these results suggested that human CAMP/LL-37 might act as a tumor-suppressor in OSCC and DNA methylation might play roles during carcinogenesis via directly downregulating human CAMP promoter activity.

Effects of local delivery of bFGF from PLGA microspheres on osseointegration around implants in diabetic rats.

OBJECTIVE: Diabetes mellitus may impair bone healing after dental implant placement. The objective of this study was to evaluate the effects of the local delivery of basic fibroblast growth factor (bFGF) from poly(lactide-co-glycolide) (PLGA) microspheres on osseointegration around titanium implants in diabetic rats. STUDY DESIGN: The bFGF-PLGA microspheres were prepared by the W/O/W double-emulsion solvent evaporation method. A total of 20 rats were used to create diabetic animal models by giving them a high-fat and high-sugar diet and a low-dose streptozotocin intraperitoneal injection. Titanium implants were planted into the tibias of the diabetic rats and into 10 normal rats. Microspheres were loaded on the surfaces of the implants in the bFGF intervention group before they were placed into the rats. After 4 or 8 weeks, the tibias containing the implants were removed and embedded with resin. Uncalcified tissue slices were prepared to compare osseointegration. RESULTS: At 4 weeks, the bone-implant contact rate in the diabetic control group was less than that in the control group and the bFGF intervention group (P < .05). At 8 weeks, the results among the 3 groups were similar to those at 4 weeks. CONCLUSIONS: The local delivery of bFGF from PLGA microspheres into areas around titanium implants may improve osseointegration in diabetic rats.

In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

Observations on the survival and neovascularization of fat grafts interchanged between C57BL/6-gfp and C57BL/6 mice.

BACKGROUND: Autologous fat transplantation has become a prevalent option for soft-tissue augmentation throughout the body. However, there is still much controversy over whether the fat grafts have survived or have been replaced in the recipient sites and over how the vessels grow. METHODS: After C57BL/6-gfp mice and C57BL/6 mice were paired randomly, the inguinal fat was excised and cut into pieces with scissors, and the adipose granules, approximately 0.2 ml (0.195 g), were transplanted subcutaneously with syringes to the dorsa of the paired mice. Samples were obtained at different time intervals: 3 days, 7 days, 2 weeks, 4 weeks, 2 months, 3 months, and 4 months after transplantation. Each fat sample was weighed to evaluate the graft volume. Histology, origin, and densities of neovascularization were examined by immunohistochemical staining. RESULTS: At 4 months, there was no significant difference in either graft survival or histologic evaluation. Histologic evaluation manifested the normal physiologic process of inflammation, neovascularization, remodeling, and maturity at different time intervals. At the endpoint, the immunohistochemical staining of CD34 showed that the difference in capillary density of the fat graft-31.3 ± 3.9 capillaries/mm on the dorsa of the C57BL/6-gfp mice and 29.6 ± 3.2 capillaries/mm on the dorsa of the C57BL/6 mice-was not statistically significant. The α-smooth muscle actin staining indicated that there were neovascularized vessels in both C57BL/6-gfp and C57BL/6 fat grafts. CONCLUSIONS: Fat grafts can survive and neovascularized vessels can grow from the recipient sites. Fat transplantation is feasible and will be applied more widely if fat graft survival is improved.

A novel role of protein tyrosine kinase2 in mediating chloride secretion in human airway epithelial cells.

Ca(2+) activated Cl(-) channels (CaCC) are up-regulated in cystic fibrosis (CF) airway surface epithelia. The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl(-) secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca(2+), which not only activates epithelial CaCCs, but also inhibits epithelial Na(+) hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl(-) secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca(2+) and Cl(-) secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells.

[Effect of local delivery of basic fibroblast growth factor 2 on osseointegration around implant in tibia of diabetic rats].

OBJECTIVE: To evaluate the effect of the local delivery of basic fibroblast growth factor 2 (bFGF-2) on the osseointegration around titanium implant of diabetic rats. METHODS: The bFGF-2-loaded poly (lactic-co-glycolic acid) microspheres were prepared by water/oil/water (W/O/W) double-emulsion solvent evaporation method. Thirty-five male SPF level Sprague Dawley rats, weighing 220-250 g and aged 9 weeks, were selected as experimental animals. Ten rats were fed with the routine diet as normal control group. The other 25 rats were made the diabetic animal model by giving high fat-sugar diet and a low dose streptozotocin (30 mg/kg) intravenously; 20 rats were made the diabetic animal model successfully. Then 20 rats were randomly divided into diabetic control group (n = 10) and bFGF-2 intervention group (n = 10). A hole was drilled in the right tibia bone of all rats, and the titanium implant treated by micro-arc oxidation surface was planted into the hole. Simultaneously, the previously prepared microspheres and blood were mixed and were loaded on the surface of the implant before it was implanted into the rats of the bFGF-2 intervention group. At 4 and 8 weeks, the tibia containing implants was harvested, embedded with resin and made undecalcified tissue slices to compare the osseointegration. RESULTS: At 4 weeks, the implants of the normal control group were surrounded by new lamellar bone with continuity; whereas the tissue around the implants of the diabetic control group contained little woven bone and some fibrous tissue; and obvious new formed bone with continuity was observed in bFGF-2 intervention group. At 8 weeks, the results of 3 groups were similar to those at 4 weeks. At 4 weeks, the percentage of bone-implant contact (BIC) in diabetic control group was significantly less than those in normal control group (P < 0.05) and in bFGF-2 intervention group (P < 0.05); the BIC in bFGF-2 intervention group was less than in normal control group, but showing no significant difference (P > 0.05). After 8 weeks, the BIC in normal control group and in bFGF-2 intervention group were significantly greater than that in diabetic control group (P < 0.05), but there was no significant difference between bFGF-2 intervention group and normal control group (P > 0.05). CONCLUSION: Local delivery of bFGF-2 around titanium implants may improve the osseointegration in diabetic rats.

Spiperone, identified through compound screening, activates calcium-dependent chloride secretion in the airway.

Cystic fibrosis (CF) is caused by mutations in the gene producing the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as a Cl(-) channel. Its dysfunction limits Cl(-) secretion and enhances Na+ absorption, leading to viscous mucus in the airway. Ca2+-activated Cl(-) channels (CaCCs) are coexpressed with CFTR in the airway surface epithelia. Increases in cytosolic Ca(2+) activate the epithelial CaCCs, which provides an alternative Cl(-) secretory pathway in CF. We developed a screening assay and screened a library for compounds that could enhance cytoplasmic Ca2+, activate the CaCC, and increase Cl(-) secretion. We found that spiperone, a known antipsychotic drug, is a potent intracellular Ca2+ enhancer and demonstrated that it stimulates intracellular Ca2+, not by acting in its well-known role as an antagonist of serotonin 5-HT2 or dopamine D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Spiperone activates CaCCs, which stimulates Cl(-) secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro and in CFTR-knockout mice in vivo. In conclusion, we have identified spiperone as a new therapeutic platform for correction of defective Cl(-) secretion in CF via a pathway independent of CFTR.

RNA interference targeted to multiple P2X receptor subtypes attenuates zinc-induced calcium entry.

A postulated therapeutic avenue in cystic fibrosis (CF) is activation of Ca(2+)-dependent Cl(-) channels via stimulation of Ca(2+) entry from extracellular solutions independent of CFTR functional status. We have shown that extracellular zinc and ATP induce a sustained increase in cytosolic Ca(2+) in human airway epithelial cells that translates into stimulation of sustained secretory Cl(-) transport in non-CF and CF human and mouse airway epithelial cells, cell monolayers, and nasal mucosa. On the basis of these studies, the Ca(2+) entry channels most likely involved were P2X purinergic receptor channels. In the present study, molecular and biochemical data show coexpression of P2X(4), P2X(5), and P2X(6) subtypes in non-CF (16HBE14o(-)) and CF (IB3-1) human bronchial epithelial cells. Other P2X receptor Ca(2+) entry channel subtypes are expressed rarely or not at all in airway epithelia, epithelial cell models from other CF-relevant tissues, or vascular endothelia. Novel transient lipid transfection-mediated delivery of small interference RNA fragments specific to P2X(4) and P2X(6) (but not P2X(5)) into IB3-1 CF human airway epithelial cells inhibited extracellular zinc- and ATP-induced Ca(2+) entry markedly in fura-2 Ca(2+) measurements and "knocked down" protein by >65%. These data suggest that multiple P2X receptor Ca(2+) entry channel subtypes are expressed in airway epithelia. P2X(4) and P2X(6) may coassemble on the airway surface as targets for possible therapeutics for CF independent of CFTR genotype.

Extracellular zinc and ATP restore chloride secretion across cystic fibrosis airway epithelia by triggering calcium entry.

Cystic fibrosis (CF) is caused by defective cyclic AMP-dependent cystic fibrosis transmembrane conductance regulator Cl(-) channels. Thus, CF epithelia fail to transport Cl(-) and water. A postulated therapeutic avenue in CF is activation of alternative Ca(2+)-dependent Cl(-) channels. We hypothesized that stimulation of Ca(2+) entry from the extracellular space could trigger a sustained Ca(2+) signal to activate Ca(2+)-dependent Cl(-) channels. Cytosolic [Ca(2+)](i) was measured in non-polarized human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Primary human CF and non-CF airway epithelial monolayers as well as Calu-3 monolayers were used to assess anion secretion. In vivo nasal potential difference measurements were performed in non-CF and two different CF mouse (DeltaF508 homozygous and bitransgenic gut-corrected but lung-null) models. Zinc and ATP induced a sustained, reversible, and reproducible increase in cytosolic Ca(2+) in CF and non-CF cells with chemistry and pharmacology most consistent with activation of P2X purinergic receptor channels. P2X purinergic receptor channel-mediated Ca(2+) entry stimulated sustained Cl(-) and HCO(3)(-) secretion in CF and non-CF epithelial monolayers. In non-CF mice, zinc and ATP induced a significant Cl(-) secretory response similar to the effects of agonists that increase intracellular cAMP levels. More importantly, in both CF mouse models, Cl(-) permeability of nasal epithelia was restored in a sustained manner by zinc and ATP. These effects were reversible and reacquirable upon removal and readdition of agonists. Our data suggest that activation of P2X calcium entry channels may have profound therapeutic benefit for CF that is independent of cystic fibrosis transmembrane conductance regulator genotype.

Sustained calcium entry through P2X nucleotide receptor channels in human airway epithelial cells.

Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing [Ca(2+)](i) in human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Cytosolic Ca(2+) was measured by fluorospectrometry using the fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in [Ca(2+)](i) derived from intracellular stores in a Na(+)-rich environment. In contrast, ATP induced an increase in [Ca(2+)](i) that had transient and sustained components in a Na(+)-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased [Ca(2+)](i) only in Na(+)-free medium, suggesting competition between Na(+) and Ca(2+) through P2XRs. Biochemical evidence showed that the P2X(4) receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca(2+) channels, voltage-dependent Ca(2+) channels, or Na(+)/Ca(2+) exchanger in the ATP-induced sustained Ca(2+) signal was ruled out. In conclusion, these data show that epithelial P2X(4) receptors serve as ATP-gated calcium entry channels that induce a sustained increase in [Ca(2+)](i). In airway epithelia, a P2XR-mediated Ca(2+) signal may have therapeutic benefit for CF.