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Purinergic effects in vivo are local, specific, transient, and affect target cells in a paracrine or autocrine manner. Purinergic signalling is involved in the regulation of numerous functions in the male and female reproductive tract organs. Analysis of functional expression of purinoceptors suggests that P2Y2 receptors are involved in the regulation of luminal fluid secretion in vivo; P2X1 and P2X2 in the regulation of smooth muscle cell contraction of blood vessels and tubular organs; P2X2 in the regulation of sperm cell maturation; P2X3 in activation of sensory nerve fibers involved in reflex activities and pain; P2X4 in the regulation of tight junctional resistance and epithelial transport in female reproductive tract epithelia, and in the regulation of luminal acidification in the epididymis, vas deferens and probably the vagina and ectocervix; P2Y1 and P2Y2 in the regulation of cell proliferation; P2X5 and P2X7A in the regulation of epithelial cell terminal differentiation and apoptosis; and A1 receptors in the regulation of sperm cell capacitation. Translational studies suggested that cellular levels of the P2X7A receptor could be used as a biomarker for the early detection of breast, ectocervix, endocervix, endometrial and bladder cancers. Data also suggested that the P2X7A mechanism could be used as a pharmaceutical target for the prevention and treatment of epithelial neoplasia through the induction of apoptosis.

The P2X7A receptor is the main physiological pro-apoptotic mechanism in epithelia in vivo. The natural ligand of the P2X7A is ATP. The P2X7A is expressed abundantly in epithelial reserve (stem) cells, and it regulates epithelial-cell terminal differentiation. The high expression of P2X7A in reserve cells renders the cells sensitive to conditions of increased extracellular ATP, in particular upon neoplastic transformation, and to the induction of apoptosis. Cellular expression of the P2X7A is reduced in cancer cells of epithelia derived from the ectoderm, the urogenital sinus, and the distal paramesonephric duct. Low levels of P2X7A have been linked to cancer development because reserve cells harboring a defective P2X7A mechanism can escape P2X7A pro-apoptotic control. Mechanisms that induce reduced expression of P2X7A in cancer epithelial cells involve hypermethylation of the P2X7 gene and decreased transcription; enhanced degradation of the P2X7 transcript through the action of micro-RNAs miR-186 and miR-150; and epidermal growth factor (EGF) regulated, protein kinase-A (PKA)-mediated de-glycosylation of the P2X7A. Reduced cellular P2X7A content has been proposed as a biomarker for human breast, bladder, ectocervix, endocervix, and endometrial cancers. The chemotherapeutic potential of augmented P2X7A-dependent apoptosis of neoplastic epithelial cells was recently demonstrated in the mouse in vivo. Local treatment on the skin with the P2X7A-specific agonist benzoyl-ATP (BzATP) decreased the development of 7,12-dimethyl-benz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin papillomas and skin cancers by 50%, by an effect that involves augmentation of keratinocytes reserve cells apoptosis. The P2X7A mechanism represents a novel pharmaceutical target for the prevention and treatment of epithelial neoplasia through the induction of apoptosis. WIREs Membr Transp Signal 2012,1:349–371. doi: 10.1002/wmts.33 For further resources related to this article, please visit the WIREs website.

Anti-apoptotic mechanisms contribute to the development of cancer. The purinergic ligand-gated ion channel receptor P2X(7) system is an important pro-apoptosis modulator in epithelial cells, and augmentation of P2X(7)-mediated apoptosis has been proposed as a novel pharmacological modality for chemoprevention and treatment of epithelial cancers.This review focuses on the progress towards clinical application of therapies that directly modulate P2X(7)-mediated apoptosis. This paper reviews and discusses P2X(7)-mediated apoptosis in the context of prevention and growth-control of epithelial cancer cells, and summarizes recent data in the mouse skin model in vivo.Data for this review were identified from the PubMed and MEDLINE databases.The growing understanding of mechanisms of P2X(7)-mediated apoptosis has generated a novel strategy for targeting directly and specifically skin neoplasia. The data link directly, for the first time, upregulation of apoptosis with cancer chemoprevention. Striking antitumor efficacy has been achieved in the rodent cancer model, and it is likely that the recently gained wealth of knowledge about the mechanisms of P2X(7)-control of apoptosis will eventually be translated into new clinical therapies for epithelial cancers.

The pro-apoptotic P2X(7) receptor regulates growth of epithelial cells. The objectives of the study were to understand P2X(7) gene transcription; to identify the active promoter and the transcription initiation site (TpIS); and to begin understanding regulation of P2X(7) gene transcription. Experiments in vitro utilized normal and cancerous cultured human uterine cervical epithelial cells, and HEK293 cells overexpressing P2X(7)-luciferase reporters. Experiments in vivo used surgical specimen of normal and cancerous uterine cervix. Assays involved DNA, RNA, and protein techniques. (a) The P2X(7) TpIS was localized to adenine (+1) at nt 1683 of the human P2X(7) gene [GenBank Y12851]), with a TTAAA sequence at nt -32/-28 and an active promoter region within nt -158/+32. (b) P2X(7) transcription was found to be regulated by two enhancers located at nt + 222/+232 and +401/+573 regions downstream of the active P2X(7) promoter. (c) The putative enhancer regions formed four DNA-protein complexes. (d) P2X(7) transcription was found to be controlled by hypermethylated cytosines at cytosine-phosphodiester-guanosines (CpG) that cluster or co-localize with the enhancers' sites. (e) We identified nine CpGs as inhibitory cis elements, and three CpG sites that are hypermethylated in cultured cervical epithelial cells and in cervix epithelia in vivo. (f) In cancer cervical cells, the degree of hypermethylation of the CpG sites was greater than in the normal cervical cells. Expression of the P2X(7) receptor is controlled by hypermethylated CpGs that flank transcription enhancers located within a 547-nt region downstream of the promoter.

The P2X7 receptor regulates cell growth through mediation of apoptosis. P2X7 levels are lower in cancer epithelial cells than in normal cells, and previous studies showed that expression of P2X7 was regulated post-transcriptionally. The objective of the study was to understand regulation of P2X7 mRNA stability. Overexpression of a reporter containing the full-length human P2X7 3′-untranslated region (3′-UTR) or reporters containing parts of the 3′-UTR-P2X7 were associated with increased abundance of the construct in normal cells and decreased abundance in cancer epithelial cells. Sequences within the 3′-UTR-P2X7, which are putative target sites for the microRNAs, miR-186 (middle segment) and miR-150 (distal segment), decreased the abundance of the P2X7 transcript. Overexpression in cancer cells of mutated miR-186 and miR-150 target sites was associated with lower levels of the reporter genes. In normal cells overexpression of the mutated miR-186 target site was associated with marked increased concentration, but overexpression of the miR-150 target site reporters, wild-type and mutant, did not change over time. Levels of miR-186 and miR-150 were higher in cancer than in normal cells, and treatment with miR-186 and miR-150 inhibitors increased P2X7 mRNA. In human embryonic kidney-293 cells heterologously expressing the full-length 3′-UTR-P2X7 luciferase reporter, miR-186 and miR-150 inhibitors increased luciferase activity, whereas miR-186 and miR-150 mimics decreased luciferase activity after actinomycin D treatment. These data suggest that increased expression of miR-186 and miR-150 in cancer epithelial cells decreases P2X7 mRNA by activation of miR-186 and miR-150 instability target sites located at the 3′-UTR-P2X7.

An important role of estrogen is regulation of epithelial permeability and the control of fluid transudation into the cervical and vaginal lumens. Our present understanding of transepithelial transport in relatively leaky types of epithelia, such as the cervical and vaginal epithelia, is based on the pioneering works of Ussing and Zerahn1 and Spring and Hope.2 The Ussing-Zerahn model predicts that luminal fluids are derived from the blood compartment; that plasma moves through the epithelial paracellular (intercellular) space (Fig. 1B), and that paracellular transport of the fluid is controlled by the resistance of the epithelial tight junctions (RTJ) and of the epithelial lateral intercellular space (RLIS). The RTJ and the RLIS contribute in series to the total resistance (RTE) so that RTE ≈ RTJ + RLIS. The RTJ is a relatively high resistance and is determined by occlusion of the intercellular space through the interactions of extracellular loops of transcellular tight junctional proteins such as the claudins3 and occludin4 (Fig. 1D-E). The RLIS is considered a low transepithelial resistance and is determined by the proximity of neighboring epithelial cells (Fig. 1A). The Spring-Hope model2 predicts that the driving force moving the fluid from the blood into the lumen is the capillary blood pressure, which creates a subluminal to luminal hydrostatic gradient (Fig. 1A and B). Subluminal to luminal hydrostatic gradients dilate the lateral intercellular space, thereby decreasing the RLIS (Fig. 1B). Dilation is restricted to the basolateral intercellular space because of the apical location of adherens intercellular junctions, which prevent lateral separation near the apical regions of the cells (Fig. 1B). FIG. 1 Models of estrogen regulation of transepithelial permeability. A: The main resistances for free diffusion of fluid from the capillary bed into the lumen through the paracellular (intercellular) space are the tight junctional resistance (RTJ) and the resistance ... Estrogen increases fluid transudation by a number of known mechanisms. Estrogen decrease in capillary resistance increases blood flow, which would tend to increase the subluminal to luminal hydrostatic gradient (Fig. 1C).5 However, this effect contributes relatively little to net fluid transudation,6,7 and the main estrogenic control of transepithelial transport is by modulation of epithelial permeability through regulation of the RTJ (Fig. 1C-E) and the RLIS (Fig. 1C, F, G). Estrogen decrease in RTJ is thought to be mediated by matrix metalloproteinase-7-dependent modulation of occludin,8-10 and the decrease in RTJ is explained by loss of gating of the intercellular space due to degradation of occludin extracellular loops.10 Estrogen decrease in the RLIS involves at least three known molecular mechanisms: actin depolymerization and the formation of monomeric-dominated G-actin cytoskeleton11; decreased dimerization of nonmuscle myosin type II-B (NMMII-B), the predominant cytoskeletal epithelial myosin12,13; and increased magnesium-adenosine triphosphatase (MgATPase) activity of the NMMII-B.14 Collectively the actin and NMMII-B effects of estrogen would tend to fragment the cortical actomyosin ring and induce formation of a dynamic cytoskeleton (Fig. 1E-G). This would facilitate dilation of the basolateral intercellular space (ie, decrease the RLIS) in response to the capillary subluminal to luminal hydrostatic gradients (Fig. 1C). The objectives of the present study were to better understand estrogen modulation of the RTJ and RLIS and how menopause affects estrogen actions. The results revealed that estrogen increases epithelial permeability by inducing an early transient decrease in RLIS followed by a slower persistent decrease in RTJ. The potency of estrogen actions was similar in cells of premenopausal and postmenopausal women, but the effects on NMMII-B filamentation and MgATPase activity were shorter in cells of postmenopausal women, resulting in faster reversal of the decrease in RLIS.

Objectives. To understand the potential role of P2X7 as biomarker of endometrial cancer, and the molecular mechanisms by which cancerous epithelial cells maintain low expression of P2X7. Methods. Feasibility clinical experimental study. Normal (28), simple or complex hyperplasia (7), complex hyperplasia with atypia (6) and cancer endometrial discarded tissues (40) were obtained from a total of 81 women, ages 25–75. Endpoint for P2X7 protein was average pixel signal density of tissue immunoreactivity with anti-P2X7 antibody. Endpoint for P2X7 mRNA was one-step quantitative Real-Time PCR. Experiments in-vitro included normal (hEVEC) and cancerous cervical epithelial cells (HeLa) transfected with reporter plasmid containing luciferase-3′ untranslated region (3′UTR)-P2X7 cDNA, using as endpoint steady-state luciferase mRNA levels. Results. Levels of P2X7 protein and mRNA were significantly lower in vivo, in tissues of complex hyperplasia with atypia or endometrial adenocarcinoma, than in tissues of normal endometrium, simple hyperplasia or complex hyperplasia tissues (sensitivity and specificity of 89– 100%, p

Estrogen modulates tight junctional resistance through estrogen receptor-alpha-mediated remodeling of occludin. The objective of the study was to understand the mechanisms involved. Experiments using human normal vaginal-cervical epithelial cells showed that human normal vaginal-cervical epithelial cells secrete constitutively matrix-metalloproteinase-7 (MMP-7) into the luminal solution and that MMP-7 is necessary and sufficient to produce estrogen decrease of tight junctional resistance and remodeling of occludin. Treatment with estrogen stimulated activation of the pro-MMP-7 intracellularly and augmented secretion of the activated MMP-7 form. Steady-state levels of MMP-7 mRNA and protein were not affected by estrogen. Estrogen modulated phosphorylation of the MMP-7, but the changes were most likely secondary to changes in cellular MMP-7 mass. Estrogen increased coimmunoreactivity of MMP-7 with the Golgi protein GPP130. Tunicamycin and brefeldin-A had no effect on cellular MMP-7 but monensin (inhibitor of Golgi traffic) blocked estrogen effects, suggesting estrogen site of action is at the Golgi system. Estrogen increased generalized secretory activity, including of luminal exocytosis of polycarbohydrates. However, estrogen increased coimmunoreactivity of MMP-7 with synaptosomal-associated protein of 25 kDa in apical membranes, suggesting soluble N-ethylmaleimide sensitive fusion factor attachment protein receptor-facilitated exocytosis of MMP-7. Treatment with the vesicular-ATPase inhibitor bafilomycin A(1) inhibited activation of MMP-7. These data suggest that estrogen up-regulates activation of the MMP-7 intracellularly, at the level of Golgi, and augments secretion of activated MMP-7 through soluble N-ethylmaleimide sensitive fusion factor attachment protein receptor-dependent exocytosis. On the other hand, estrogen acidification of the luminal solution would tend to alkalinize exocytotic vesicles and may lead to decreased activation of the MMP-7. These mechanisms acting in concert could be important for regulation and control of estrogen modulation of paracellular permeability in vivo.

The study tested the hypothesis that estrogen controls epithelial paracellular resistance through modulation of myosin. The objective was to understand how estrogen modulates nonmuscle myosin-II-B (NMM-II-B), the main component of the cortical actomyosin in human epithelial cervical cells. Experiments used human cervical epithelial cells CaSki as a model, and end points were NMM-II-B phosphorylation, filamentation, and MgATPase activity. The results were as follows: 1) treatment with estrogen increased phosphorylation and MgATPase activity and decreased NMM-II-B filamentation; 2) estrogen effects could be blocked by antisense nucleotides for the estrogen receptor-alpha and by ICI-182,780, tamoxifen, and the casein kinase-II (CK2) inhibitor, 5,6-dichloro-1-beta-(D)-ribofuranosylbenzimidazole and attenuated by AG1478 and PD98059 (inhibitors of epithelial growth factor receptor and ERK/MAPK) but not staurosporine [blocker of protein kinase C (PKC)]; 3) treatments with the PKC activator sn-1,2-dioctanoyl diglyceride induced biphasic effect on NMM-II-B MgATPase activity: an increase at 1 nm to 1 microM and a decrease in activity at more than 1 microM; 4) sn-1,2-dioctanoyl diglyceride also decreased NMM-II-B filamentation in a monophasic and saturable dose dependence (EC(50) 1-10 microM); 5) when coincubated directly with purified NMM-II-B filaments, both CK2 and PKC decreased filamentation and increased MgATPase activity; 6) assays done on disassembled NMM-II-B filaments showed MgATPase activity in filaments obtained from estrogen-treated cells but not estrogen-depleted cells; and 7) incubations in vitro with CK2, but not PKC, facilitated MgATPase activity, even in disassembled NMM-II-B filaments. The results suggest that estrogen, in an effect mediated by estrogen receptor-alpha and CK2 and involving the epithelial growth factor receptor and ERK/MAPK cascades, increases NMM-II-B MgATPase activity independent of NMM-II-B filamentation status.

To understand myosin regulation of epithelial permeability.This was an experimental study, using human cervical epithelial cells CaSki. End points were paracellular permeability (determined in terms of transepithelial electrical resistance); non-muscle myosin-II-B (NMM-II-B) cellular localization; NMM-II-B phosphorylation status; NMM-II-B-actin interaction (determined in vitro by the immunoprecipitation-immunoreactivity method); and NMM-II-B filamentation (determined in vitro using purified NMM-II-B filaments in terms of filaments disassembly/assembly ratios.Treatment of cells with the Rho-associated kinase (ROCK) inhibitor Y-27632 or with the phosphatase inhibitor okadaic acid decreased the resistance of the lateral intercellular space (R(LIS)), and increased phosphorylation of NMM-II-B on threonine and serine residues. Y-27632 induced disorganization of the cortical acto-myosin and decreased co-immunoprecipitation of actin with NMM-II-B. Homodimerization assays using NMM-II-B filaments from cells treated with Y-27632 or okadaic acid revealed decreased filamentation compared to control cells. However, okadaic acid blocked Y-27632 decreased filamentation. Treatment with DRB, a casein kinase-II (CK2) inhibitor, induced opposing effects to those of Y-27632 and okadaic acid. Treatment with 5,6-dichloro-1-beta-(D)-ribofuranosylbenzimidazole (DRB) did not involve modulation of actin depolymerization, suggesting that NMM-II-B regulation of the R(LIS) was independent of actin polymerization status. Exposure of NMM-II-B filaments to CK2 increased filamentation, regardless of prior treatments in vivo with Y-27632, okadaic acid, or DRB.The results suggest that NMM-II-B filaments are in steady-state equilibrium of phosphorylation-dephosphorylation mediated by CK2 and by ROCK-regulated myosin heavy chain phosphatase, respectively. Increased phosphorylation would tend to inhibit assembly of NMM-II-B filaments and lead to decreased actin-myosin interaction, which would tend to decrease the R(LIS) and increase the paracellular permeability.

Objective: To determine expression of the P2X7 receptor in normal and in cancer uterine tissues. The rationale was that the receptor P2X7 regulates constitutive apoptosis in uterine epithelial cells, and previous studies showed diminished P2X7-mediated apoptosis in cancer uterine cells compared with normal cells. Methods: A clinical, experimental feasibility study. Normal (n = 42) and cancer uterine tissues (n = 47) were obtained from a total of 72 women ages 25 to 75. End points for P2X7 mRNA were quantitative PCR and in situ hybridization, and end points for P2X7 protein were Western blots and immunostaining using anti-P2X7 antibody. Results: (a) In normal uteri, P2X7 mRNA and protein were expressed predominantly in the epithelial (endometrial, endocervical, and ectocervical) cells. (b) Expression of the P2X7 mRNA and protein was absent from endometrial and endocervical adenocarcinoma tissues and from cervical squamous cell carcinoma tissues. (c) In cervical dysplasia, P2X7 protein was absent in the dysplastic lesions. (d) Semiquantitative analysis using P2X7 mRNA (normalized in each tissue to the constitutive glyceraldehyde-3-phosphate dehydrogenase) and P2X7 protein levels (normalized in each tissue to the constitutive tubulin) revealed that P2X7 mRNA and/or protein levels can distinguish uterine normal from cancer tissues at high degrees of sensitivity (92%, 100%) and specificity (100%, 90%). Summary and Conclusions: (a) Levels of the P2X7 are lower in uterine epithelial cancer tissues than in the corresponding normal tissues. (b) The data suggest that tissue P2X7 mRNA and protein levels could be used as a novel biomarker to differentiate normal and cancer uterine epithelial tissues. (Cancer Epidemiol Biomarkers Prev 2006;15(10):1906–13)

Treatment of human epithelial cervical cells CaSki attached on filters with the P2X7-receptor (P2X7-R) agonist BzATP induced acute transient influx of calcium, most likely the result of P2X7-R channel activation, followed by slower sustained calcium influx. In cultures incubated in the presence of ethidium bromide (EB), BzATP induced slow and sustained influx of the dye with a time-course similar to the late slow calcium influx, suggesting P2X7-R pore formation. The acute and late calcium effects of BzATP were greater if the agonist was added to the luminal solution, facing the apical membrane of the cells. The EB effect of BzATP initially occurred in the apical membrane, while effects in the basolateral membrane were delayed and smaller. These results suggest that in polarized epithelial cells under steady-state conditions the P2X7-R is located in the apical membrane, and activation of the receptor induces formation of P2X7-R pores preferentially in the apical membrane.

The objective of this study was to understand the mechanisms involved in P2X7receptor activation. Treatments with ATP or with the P2X7receptor-specific ligand 2′,3′- O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) induced pore formation, but the effect was slower in CaSki cells expressing endogenous P2X7receptor than in human embryonic kidney (HEK)-293 cells expressing exogenous P2X7receptor (HEK-293-hP2X7-R). In both types of cells Western blots revealed expression of three forms of the receptor: the functional 85-kDa form present mainly in the membrane and 65- and 18-kDa forms expressed in both the plasma membrane and the cytosol. Treatments with ATP transiently decreased the 85-kDa form and increased the 18-kDa form in the membrane, suggesting internalization, degradation, and recycling of the receptor. In CaSki cells ATP stimulated phosphorylation of the 85-kDa form on tyrosine and serine residues. Phosphorylation on threonine residues increased with added ATP, and it increased ATP requirements for phosphorylation on tyrosine and serine residues, suggesting a dominant-negative effect. In both CaSki and in HEK-293-hP2X7-R cells ATP also increased binding of the 85-kDa form to G protein-coupled receptor kinase (GRK)-3, β-arrestin-2, and dynamin, and it stimulated β-arrestin-2 redistribution into submembranous regions of the cell. These results suggest a novel mechanism for P2X7receptor action, whereby activation involves a GRK-3-, β-arrestin-2-, and dynamin-dependent internalization of the receptor into clathrin domains, followed in part by receptor degradation as well as receptor recycling into the plasma membrane.

The objective of the study was to understand age-related contributions of the resistance of the intercellular tight junctions (R(TJ)) and the resistance of the lateral intercellular space (R(LIS)) to the transcervical-transvaginal permeability. The experiments used normal human ectocervical epithelial cells obtained from women aged 36-65 yr. Twenty-four hours of treatment of cells with 10 nm 17beta-estradiol decreased the R(LIS), whereas longer treatments were required to decrease The R(TJ). Aging had no effect on baseline or estrogen decrease in R(TJ), but it blocked both baseline and the estrogen decrease in R(LIS). Actin assays showed age-related decrease in the fraction of monomeric G-actin and attenuation of sodium-nitroprusside-induced increase in G-actin. These results suggest that the aging-related diminished capacity of human ectocervical epithelial cells to remodel cellular actin from polymerized actin toward monomeric G-actin confers increased cell rigidity and therefore increased R(LIS). Therefore, the hypoestrogenism-related decrease in R(TJ) and the hypoestrogenism- and aging-related increase in R(LIS) could be the cellular mechanisms of decreased permeability that lead to decreased fluid transport and decreased lubrication of the lower genital tract in older postmenopausal women.

Epidermal growth factor (EGF), epinephrine, and the P2X7 receptor system regulate growth of human uterine cervical epithelial cells, but little is known about how these systems intercommunicate in exerting their actions. The objective of this study was to understand the mechanisms of EGF and epinephrine regulation of growth of cervical cells. Treatment of cultured CaSki cells with 0.2 nM EGF increased cell number via a PD98059-sensitive pathway. Treatment with 2 nM epinephrine increased cell number, and the effect was facilitated by cotreatment with EGF. Whereas the effect of EGF alone involved up-regulation of [3H]-thymidine incorporation and an increase in cell proliferation, the effect of epinephrine was mediated by inhibition of apoptosis. Epinephrine inhibited apoptosis induced by the P2X7 receptor ligand 2',3'-0-(4-benzoylbenzoyl)-ATP, by attenuation of P2X7 receptor plasma membrane pore formation. Cotreatment with EGF facilitated epinephrine effect via a phosphoinositide 3-kinase-dependent mechanism. CaSki cells express the beta2-adrenoceptor, and the epinephrine antiapoptotic effect could be mimicked by beta2-adrenoceptor agonists and by activators of adenylyl cyclase. Likewise, the effect could be blocked by beta2-adrenoceptor blockers and by the inhibitor of protein kinase-A H-89. Western immunoblot analysis revealed that epinephrine decreased the levels of the glycosylated 85-kDa form of the P2X7 receptor and increased receptor degradation, and that EGF potentiated these effects of epinephrine. EGF did not affect cellular levels of the beta2-adrenoceptor. In contrast, EGF, acting via the EGF receptor, augmented beta2-adrenoceptor recycling, and it inhibited beta2-adrenoceptor internalization via a phosphoinositide 3-kinase-dependent mechanism. We conclude that, in cervical epithelial cells, EGF has a dual role: as mitogen, acting via the MAPK/MAPK kinase pathway, and as an antiapoptotic factor by facilitating epinephrine effect and resulting in greater expression of beta2-adrenoceptors in the plasma membrane. These findings underscore a novel signaling network of communication between the receptor tyrosine kinases, the G protein-coupled receptors, and the purinergic P2X7 receptor.

The present study investigated the antiapoptotic effects of estrogen in normal and cancer human cervical cells and the mechanisms involved. Baseline apoptosis in human cervical epithelial cells is mediated predominantly by P2X7-receptor-induced, Ca2+-dependent activation of the mitochondrial (caspase-9) pathway. Treatment with 10 nm 17β-estradiol blocked apoptosis induced by the P2X7-receptor ligands ATP and 2′,3′-0-(4-benzoylbenzoyl)-ATP in normal human cervical epithelial cells (hECEs) and attenuated the effect in hECEs immortalized with human papillomavirus-16 (ECE16–1) and the cancer cervical cells HT3 and CaSki. Diethylstilbestrol and to a lesser degree estrone could mimic the effects of 17β-estradiol, whereas actinomycin-D and cycloheximide attenuated the response. The antiapoptotic effect of estrogen did not depend on cell cycle phase, and in both normal and cancer cervical cells, it involved attenuation of activation of caspase-9 and the terminal caspase-3. However, involvement of cascades upstream to the caspase-9 differed in normal vs. cancer cervical cells. In the normal hECEs estrogen blocked P2X7-receptor-induced calcium influx. In contrast, in the cancer CaSki cells, estrogen up-regulated expression of Bcl-2 and attenuated Ca2+-induced mitochondrial swelling (i.e. formation of mitochondrial permeability transition pores). Estrogen had no effect on P2X7-receptor-induced apoptosis in the anaplastic SiHa and Hela cells. These results point to a novel antiapoptotic effect of estrogen in the cervix that is independent of its mitogenic function. The results also suggest that cancer cervical cells evolved antiapoptotic mechanisms that enable the cells to evade apoptosis and could therefore promote tumor progression.

This paper reviews new models of nitric-oxide (NO)-related regulation of transepithelial and transendothelial permeability in normal tissues, with emphasis on physiological relevance of the data. Novel data obtained in cultured human epithelial and endothelial cells indicate that NO can originate from different intracellular sources, and can be selectively regulated and employed to activate various intracellular mechanisms that will affect the permeability via different mechanisms. These models may be important for understanding NO regulation of permeability in vivo.

Cardiovascular disease (CVD), and in particular coronary artery heart disease (CAHD), is the leading cause of morbidity and mortality in women. Until recently, most of our knowledge about the pathophysiology of CVD in women - and, subsequently, management guidelines - were based on studies conducted mostly in men. While similar mechanisms operate to induce CVD in women and men, gender-related differences exist in the anatomy and physiology of the myocardium, and sex hormones modify the course of disease in women. Women, more than men, have their initial manifestation of CAHD as angina pectoris; are likely to be referred for diagnostic tests at a more advanced stage of disease, and are less likely than men to have corrective invasive procedures. The overall morbidity and mortality following the initial ischaemic heart event is worse in women, and the case fatality rate is greater in women than in men. Also, the relative impact of impaired vasoreactivity of the coronary artery, increased viscosity of the blood and dysregulation of automaticity and arrhythmia, is greater in women than in men. The most effective means of decreasing the impact of CVD on women's health is by an active approach from childhood to proper principles of healthcare in order to modify the contribution of specific risk factors. The latter include obesity, abnormal plasma lipid profile, hypertension, diabetes mellitus, cigarette smoking, sedentary lifestyle, increased blood viscosity, augmented platelet aggregability, stress and autonomic imbalance. The use of lipid-lowering drugs has not been adequately studied in women but reports from studies conducted mostly in men do predict an advantage also to women. Oestrogen deficiency after spontaneous or medically induced menopause is an important risk factor for CVD and CAHD. Observational and mechanistic data suggest a role for oestrogen replacement after menopause for primary, and possibly secondary, prevention of CVD. However, two recent prospective trials suggest that treatment de novo with hormone replacement of older post-menopausal women after an acute coronary event may not confer cardiovascular protection and may increase the risk of thromboembolic disease. Results of ongoing long-term studies may determine the beneficial role of hormone replacement versus potential risks involved with this treatment.

Micromolar concentrations of ATP stimulate biphasic change in transepithelial conductance across CaSki cultures, an acute increase (phase I response) followed by a slower decrease ( phase II response). Phase I and phase II responses involve two distinct calcium-dependent pathways, calcium mobilization and calcium influx. To test the hypothesis that phase I and phase II responses are mediated by distinct P2 purinergic receptors, changes in permeability were uncoupled by blocking calcium mobilization with 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA) or by lowering extracellular calcium, respectively. Under these conditions ATP EC50was 25 μM for phase Iresponse and 2 μM for phase II response. The respective agonist profiles were ATP > UTP > adenosine 5′- O-(3-thiotriphosphate) (ATP-γS) = N6-([6-aminohexyl]carbamoylmethyl)adenosine 5′-triphosphate (A8889) > GTP and UTP > ATP > GTP = A8889 > ATP-γS. Suramin blocked phase Iresponse and ATP-induced calcium mobilization, whereas pyridoxal phosphate-6-azophenyl-2′,4-disulfonic acid (PPADS) blocked phase II response and ATP-augmented calcium influx. ATP time course and pharmacological profiles for phase II response and augmented calcium influx were similar, with a time constant of 2 min and a saturable concentration-dependent effect (EC50of 2–3 μM). RT-PCR experiments revealed expression of mRNA for both the P2Y2and P2X4receptors. These results suggest that the ATP-induced phase I and phase IIresponses are mediated by distinct P2 purinergic receptor mechanisms.

The objective of the study was to understand effects of estrogen and aging on paracellular permeability of human vaginal-cervical epithelia. Assays included determinations of transepithelial electrical conductance across cultures of normal human ectocervical epithelial cells on filters. Baseline transepithelial electrical conductance across steroid-deprived cells from postmenopausal women was lower than across cells of premenopausal women. Short-term (24-48 h) treatment with 10 nM 17beta-estradiol increased transepithelial electrical conductance in both groups of cells. In cells of premenopausal women longer-term treatment with estrogen for up to 14 d had no additional effect on permeability, but in cells of postmenopausal women it caused a secondary increase in transepithelial electrical conductance that continued for the duration of the 2-wk treatment. Binding assays of 17beta-[(3)H]estradiol revealed saturable binding to high affinity (1.2-1.3 nM), low capacity sites (0.2-1.2 pmol/mg DNA) in cells of both premenopausal and postmenopausal women. In both types of cells treatment with 17beta-estradiol increased 17beta-[(3)H]estradiol binding activity in a time- and dose-related manner (EC(50) 1 nM; maximal effect within 9-12 h), and increased estrogen receptor-alpha and -beta mRNA. 8-Br-cGMP, a stable cell-permeant analog of cGMP, could mimic the estrogen first phase increase in transepithelial electrical conductance, but not the secondary increase. Treatment with estrogen augmented the increase in transepithelial electrical conductance that was induced by hydrostatic gradients (modulator of the resistance of the lateral intercellular space), and the effect was independent of woman's age or baseline transepithelial electrical conductance. In contrast, the effect of low extracellular calcium (modulator of the tight junctional resistance) was more potent in cells of premenopausal women than in cells of postmenopausal women and was independent of treatment with estrogen. These results suggest that changes in vaginal-cervical epithelial permeability after menopause are determined by aging-related increase in tight junctional resistance, and by low estrogen-dependent increase in lateral intercellular space that lead to net increase in total paracellular resistance and decreased permeability and result in reduced lubrication of the lower genital canal.

Estrogen increases baseline transepithelial permeability across CaSki cultures and augments the increase in permeability in response to hypertonic gradients. In estrogen-treated cells, lowering cytosolic calcium abrogated the hypertonicity-induced augmented increase in permeability and decreased baseline permeability to a greater degree than in estrogen-deprived cells. Steady-state levels of cytosolic calcium in estrogen-deprived cells were higher than in estrogen-treated cells. Increases in extracellular calcium increased cytosolic calcium more in estrogen-deprived cells than in estrogen-treated cells. However, in estrogen-treated cells, increasing cytosolic calcium was associated with greater increases in permeability in response to hypertonic gradients than in estrogen-deprived cells. Lowering cytosolic calcium blocked the estrogen-induced increase in nitric oxide (NO) release and in the in vitro conversion of l-[3H]arginine to l-[3H]citrulline. Treatment with estrogen upregulated mRNA of the NO synthase isoform endothelial nitric oxide synthase (eNOS). These results indicate that cytosolic calcium mediates the responses to estrogen and suggest that the estrogen increase in permeability and the augmented increase in permeability in response to hypertonicity involve an increase in NO synthesis by upregulation of the calcium-dependent eNOS.

The objective of the study was to characterize the effect and explore the mechanisms by which menopause affects paracellular permeability of cultured human cervical epithelium. The experimental system was cultures of human ectocervical epithelial (hECE) cells on filters. Assays included flux measurements of cell-impermeable molecules and determinations of transepithelial electrical conductance. hECE cells of postmenopausal women formed epithelia with lower paracellular permeability than hECE cells of premenopausal women. Treatment with estrogen increased paracellular permeability, but levels remained lower compared to cultures from premenopausal women. Lowering extracellular calcium or treatment with cytochalasin-D, conditions that decrease the tight junctional resistance (R(TJ)), increased the permeability, and the relative effects were greater in cells of premenopausal women than in postmenopausal women. Treatment of cells with sn-1,2-dioctanoyl diglyceride, an agent that increases the R(TJ), decreased the permeability, and the relative effect was greater in cells of postmenopausal women than in cells of premenopausal women. Estrogen had no significant effect on the responses to low calcium, cytochalasin-D, or sn-1,2-dioctanoyl diglyceride. Hydrostatic and hypertonic gradients in the subluminal to luminal direction, conditions that decrease the resistance of the lateral intercellular space increased the permeability to a similar degree in cultures of cells from premenopausal and from postmenopausal women. Pretreatment with estrogen augmented the increases in permeability in response to hydrostatic and hypertonic gradients. In cells exposed to low extracellular calcium, hydrostatic gradients had an additive increase in permeability. By extrapolation it was determined that in cultures of postmenopausal women R(TJ) contributes 97% to the total paracellular resistance, whereas in hECE cultures of premenopausal women the R(TJ) contributes only 84%. These results indicate that after menopause the transcervical paracellular permeability decreases significantly; this can explain the decrease in lubrication of the cervix and vagina in postmenopausal women. Part of the effect is due to lack of estrogen, and it can be reversed by treatment with the hormone. However, most of the effect is unrelated to estrogen and is caused by an increase in R(TJ).

Human cervical epithelial cells express mRNA for the nitric oxide (NO) synthase (NOS) isoforms ecNOS, bNOS, and iNOS and release NO into the extracellular medium. NG-nitro-l-arginine methyl ester (l-NAME), an NOS inhibitor, and Hb, an NO scavenger, decreased paracellular permeability; in contrast, the NO donors sodium nitroprusside (SNP) and N-(ethoxycarbonyl)-3-(4-morpholinyl)sydnonimine increased paracellular permeability across cultured human cervical epithelia on filters, suggesting that NO increases cervical paracellular permeability. The objective of the study was to understand the mechanisms of NO action on cervical paracellular permeability. 8-Bromo-cGMP (8-BrcGMP) also increased permeability, and the effect was blocked by KT-5823 (a blocker of cGMP-dependent protein kinase), but not by LY-83583 (a blocker of guanylate cyclase). In contrast, LY-83583 and KT-5823 blocked the SNP-induced increase in permeability. Treatment with SNP increased cellular cGMP, and the effect was blocked by Hb and LY-83583, but not by KT-5823. Neither SNP nor 8-BrcGMP had modulated cervical cation selectivity. In contrast, both agents increased fluorescence from fura 2-loaded cells in the Ca2+-insensitive wavelengths, indicating that SNP and 8-BrcGMP stimulate a decrease in cell size and in the resistance of the lateral intercellular space. Neither SNP nor 8-BrcGMP had an effect on total cellular actin, but both agents increased the fraction of G-actin. Hb blocked the SNP-induced increase in G-actin, and KT-5823 blocked the 8-BrcGMP-induced increase in G-actin. On the basis of these results, it is suggested that NO acts on guanylate cyclase and stimulates an increase in cGMP; cGMP, acting via cGMP-dependent protein kinase, shifts actin steady-state toward G-actin; this fragments the cytoskeleton and renders cells more sensitive to decreases in cell size and resistance of the lateral intercellular space and, hence, to increases in permeability. These results may be important for understanding NO regulation of transcervical paracellular permeability and secretion of cervical mucus in the woman.

Treatment of cultured human cervical epithelia on filters with 17beta-estradiol increases paracellular permeability in a time- and dose-related manner (EC50, 1.1 nM). The objective of the present study was to understand the molecular mechanisms of estrogen action. In cultured human cervical epithelial cells the nitric oxide (NO) donors sodium nitroprusside (SNP) and N-[ethoxycarbonyl]-3-[4-morpholinyl]sydnoneimine (SIN-I) and the cell-permeable cGMP analog 8-bromo-cGMP (8-Br-cGMP) increased paracellular permeability. In estrogen-treated cells SNP and 8-Br-cGMP increased permeability to a lesser degree than in estrogen-deprived cells, suggesting that NO and cGMP mediate the effect of estrogen on permeability. Tamoxifen blocked the estrogen-induced increase in permeability, but it had no effect on increases in permeability that were induced by SNP or by 8-Br-cGMP. LY-83583 (blocker of guanylate cyclase) attenuated the effect of SNP, whereas KT-5823 (blocker of cGMP-dependent protein kinase) abrogated the effects of both SNP and 8-Br-cGMP. Treatment with 17beta-estradiol increased NO release and cellular cGMP in a dose-related manner (EC50, approximately 1 nM), and the effects were inhibited by tamoxifen. Treatment with SNP increased cGMP maximally, even in estrogen-deficient cells. LY-83583 blocked the estrogen-induced increase in cGMP, but neither LY-83583 nor KT-5823 had a significant effect on the estrogen-induced increases in NO release and cellular cGMP. The NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester decreased NO release, and pretreatment of cells with L-arginine reversed the effect. Cultured human cervical epithelial cells express messenger RNA for the NOS isoforms endothelial NOS (ecNOS), brain NOS, and inducible NOS. 17beta-Estradiol up-regulated ecNOS messenger RNA, and tamoxifen blocked the effect. Based on these results we suggest that the effect of estradiol on permeability involves four signaling steps: 1) activation of estrogen receptors, 2) increase in ecNOS transcription and up-regulation of NO activity, 3) NO activation of guanylate cyclase and increase in cGMP, and 4) cGMP activation of cGMP-dependent protein kinase.

Estrogen increases the permeability of cultured human cervical epithelia (Gorodeski, GI. Am J Physiol Cell Physiol 275: C888-C899, 1998), and the effect is blocked by the estrogen receptor modulators ICI-182780 and tamoxifen. The objective of the study was to determine involvement of estrogen receptor(s) in mediating the effects on permeability. In cultured human cervical epithelial cells estradiol binds to high-affinity, low-capacity sites, in a specific and saturable manner. Scatchard analysis revealed a single class of binding sites with a dissociation constant of 1.3 nM and binding activity of approximately 0.5 pmol/mg DNA. Estradiol increased the density of estrogen-binding sites in a time- and dose-related manner (half time approximately 4 h, and EC(50) approximately 1 nM). RT-PCR assays revealed the expression of mRNA for the estrogen receptor alpha (alphaER) and estrogen receptor beta (betaER). Removal of estrogen from the culture medium decreased and treatment with estrogen increased the expression of alphaER and betaER mRNA. In cells not treated with estrogen, ICI-182780 and tamoxifen increased betaER mRNA. In cells treated with estrogen, neither ICI-182780 nor tamoxifen had modulated significantly the increase in alphaER or betaER mRNA. The transcription inhibitor actinomycin D blocked the estrogen-induced increase in permeability, and it abrogated the estradiol-induced increase in estrogen binding sites. These results suggest that the estrogen-dependent increase in cervical permeability is mediated by an alphaER-dependent increase in transcription.

In human cervical (CaSki) cells, extracellular adenosine triphosphate (ATP) induces an acute decrease in the resistance of the lateral intercellular space (RLIS), phase I response, followed by an increase in tight junctional resistance (RTJ), phase II response. ATP also stimulates release of calcium from intracellular stores, followed by augmented calcium influx, and both effects have similar sensitivities to ATP (EC50 of 6 microM). The objective of the study was to determine the degree to which the changes in [Ca2+]i mediate the responses to ATP. 1,2-bis (2-aminophenoxy) ethane-N,N,N1,N1-tetraacetic acid (BAPTA) abrogated calcium mobilization and phase I response; in contrast, nifedipine and verapamil inhibited calcium influx and attenuated phase II response. Barium, La3+, and Mn2+ attenuated phase I response and attenuated and shortened the ionomycin-induced phase I-like decrease in RLIS, suggesting that store depletion-activated calcium entry was inhibited. Barium and La3+ also inhibited the ATP-induced phase II response, but Mn2+ had no effect on phase II response, and in the presence of low extracellular calcium it partly restored the increase in RTJ. KCl-induced membrane depolarization stimulated an acute decrease in RLIS and a late increase in RTJ similar to ATP, but only the latter was inhibited by nifedipine. KCl also induced a nifedipine-sensitive calcium influx, suggesting that acute increases in [Ca2+]i, regardless of mobilization or influx, mediate phase I response. Phase II-like increases in RTJ could be induced by treatment with diC8, and were not affected by nifedipine. Biphasic, ATP-like changes in RTE could be induced by treating the cells with ionomycin plus diC8. We conclude that calcium mobilization mediates the early decrease in RLIS, and calcium influx via calcium channels activates protein kinase C and mediates the late increase in RTJ.

In human cervical cells, extracellular ATP induces an acute decrease in the resistance of the lateral intercellular space, the phase I response, followed by a delayed increase in tight junctional resistance, the phase II response. These responses depend on vitamin A because incubation of cells in retinoid-free medium (RFM) abolished both responses. Treatment with retinoic acid restored the phase I response in full, but the amplitude of the phase II response was restored only partly. Shorter incubations and lower concentrations of retinoic acid [half-maximal effective concentration ( K½) = 0.1 μM] were required for restoring the phase I response than were required for reversing the phase II response ( K½= 1 μM). The phase I response could be restored by ligands that bind to either retinoic acid receptors (RARs) or retinoid X receptors, but only RAR agonists had an effect on phase II response. RFM had no effect on decreases in resistance induced by ionomycin, but it attenuated phase II-like increases in resistance induced by KCl or by 1,2-dioctanoyl- sn-diglycerol (diC8). Actinomycin D blocked phase IIresponse but not phase I response or the responses to ionomycin, KCl, or diC8. These results suggest that retinoids act on cervical cells via distinct retinoid receptor mechanisms and modulate phase I and phase II changes in resistance by regulating distinct signal mechanisms.

Extracellular ATP stimulates a biphasic change in transepithelial electrical resistance (R TE ) across cultures of human cervical epithelial cells: an acute decrease (phase I), followed by a delayed increase in resistance (phase II). The objective of this study was to determine the contributions of changes in the lateral intercellular space resistance (R LIS ) and the tight junctional resistance (R TJ ) to the changes in R TE . Phase I and phase II effects were uncoupled by treatment with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester, which blocks the ATP-induced increases in cytosolic Ca 2+ and abolishes phase I. BAPTA-loaded cells differed from control cells in that 1) phase I began when ATP was added, in contrast to a delay of 1.5-3.5 min in phase II, 2) phase I decreases in R LIS followed a simple exponential pattern, in contrast to the complex kinetics of phase II, and 3) the magnitude of phase II varied between 20 and 100% for increases of R TJ in day 2-6 cultures; the phase I decrease of 50% in R LIS was unrelated to different experimental conditions. These results indicate that phase I and phase II are induced simultaneously and independently by ATP, and they contribute to the total changes in R TE . We conclude that ATP regulation of R LIS and R TJ may be important mechanisms of modulating cervical mucus production in vivo.

Estrogen or its vehicle was given daily to three groups of ovariectomized rabbits for various lengths of time, after which coronary flow was measured in their isolated perfused hearts. In one group, intramuscular injections of estrogen (40 micrograms/kg) for 7 or 14 days increased coronary flow by 40-50% (P < 0.05). In rabbits given estrogen intramuscularly for 7 days, the coronary flow returned to the basal level within 7 days after the estrogen injections were discontinued. In a second group of animals, intravenous injections of estrogen (10 micrograms/kg) for 4 days increased the coronary flow by 45% (P < 0.01). In a third group, we administered the estrogen transdermally for 4 days, and we measured the plasma estrogen levels at the end of this period. The coronary flow in this group was increased by 52% (P < 0.001), and the plasma estrogen levels ranged from 39 to 800 pg/ml. In all groups of experiments, the increments in coronary flow evoked by estrogen were virtually abolished by NG-nitro-L-arginine, an inhibitor of nitric oxide synthase. We conclude that estrogen regulates coronary blood flow, in part by upregulating nitric oxide synthase in the coronary vasculature.

Objective:To determine the effects of tamoxifen on the growth and differentiation of normal human cervical cells and compare those effects with those of a synthetic estrogen, diethylstilbestrol (DES). In addition, the effects of these compounds on immortalized cervical cells and cervical tumor cells were ascertained.Methods:Growth curves were used to determine the effects on cell proliferation. The expression of several proteins was used to determine the effects on cell differentiation. Binding assays and Western analysis were used to determine estrogen receptor levels.Results:Both tamoxifen and DES inhibited the proliferation of normal cervical cells. This growth inhibition was coincident with an increase in cell differentiation as determined by cornified envelope formation. The increase in envelope number was not accompanied by an increase in involucrin or cornifin, two protein precursors of the envelope. The activity of transglutaminase, which enzymatically incorporates precursor proteins into the enve...

Extracellular nucleotides induce a biphasic change in the transepithelial electrical conductance (GT) of human cervical cells grown on filters: a rapid increase (phase I) followed by a sustained decrease (phase II). To probe the involvement of the intercellular space, its magnitude was varied by manipulating cell volume through changes in extracellular osmolarity. Under baseline conditions [GT = 115 mS/cm2 (approximately 9 omega.cm2)] and during phase II, hypertonic challenges resulted in an increase in GT (0.98% .mosmol-1.l-1 and 0.73%.mosmol-1.l-1, respectively). However, a hypertonic challenge during phase I decreased GT (-0.16%.mosmol-1.l-1). Hypotonic challenges decreased GT during baseline, phase I, and phase II conditions by -1%.mosmol-1.l-1. Similar trends were observed with regard to pyranine permeability. Reduction of extracellular calcium increased GT, abrogated the phase II effect of extracellular ATP, and reversed the effect of a hypertonic challenge. The additive nature of the permeability changes in response to osmotic challenges and to ATP during phase II suggests that different sites are involved in each response, i.e., the resistance of the intercellular space changes with osmolarity and that of the tight junction during phase II.

OBJECTIVE We sought to determine what type of regulation of transepithelial transport in leaky epithelium can occur at the level of paracellular permeability. METHODS The epithelial permeability to the polar acid pyranine (Ppyr) and the transepithelial electrical resistance (R) were determined in cultured human cervical epithelium. RESULTS Extracellular adenosine triphosphate (ATP) acutely and reversibly decreased the paracellular permeability, as evidenced by an increase of R from 10 +/- 3 to 16 +/- 2 omega/cm2 and a decrease in Ppyr from 13 +/- 3 to 9 +/- 3 x 10(-6) cm/sec and (P < .01 for both). The ATP effect was dose related (average median effective concentration 2 +/- 1 microM), saturable at 50 microM, and desensitized with repeated administration; it was mimicked by uridine triphosphate and ATP-gamma-S but not by adenosine monophosphate, adenine, adenosine, or adenosine diphosphate. The ATP effect on resistance remained intact even when the intercellular resistance was decreased with a basal-to-apical pressure gradient, but was abolished by lowering extracellular calcium. CONCLUSIONS These results indicate the following: 1) Paracellular permeability in the cervical epithelial cell line is regulated by a nucleotide receptor; and 2) the tight junctions are effectors of nucleotide-receptor stimulation. We suggest that extracellular ATP may regulate mucus production in the cervix in vivo by acting on a surface receptor and by increasing the resistance of the tight junctions.

Transforming growth factor beta (TGF beta) is a pluripotent modulator of cell function and an important suppressor of cervical epithelial cell proliferation. In the present study, we examine the effects of TGF beta 1 on the level and activity of the epidermal growth factor receptor (EGFR) in HPV-16 immortalized cervical epithelial cells. In ECE16-1 cells, increased EGFR levels are observed within 24 h after initiation of TGF beta 1 treatment and levels continue to increase with time. This increase is correlated with a TGF beta 1-dependent decrease in proliferation rate. Scatchard analysis indicates that the population of EGFR sites induced by TGF beta 1 have a low affinity for EGF (Kd = 4.08 nM) compared to the receptors present prior to TGF beta 1 treatment (Kd = 0.3 and 1.6 nM). TGF beta 1 treatment also reduces EGFR kinase autophosphorylation activity. Cell cycle studies indicate that TGF beta 1-treated cells arrest in the G1 phase of the cell cycle and that regulation of EGFR level was independent of cell cycle stage in both TGF beta 1-treated and untreated cells. However, EGFR level was related to the G1 phase time. Parallel studies indicate that a TGF beta 1-dependent increase in p53 level is also correlated with increased time spent in G1. These results suggest that TGF beta 1 inhibition of ECE16-1 cell proliferation may act both by the replacement of high affinity/high kinase activity EGFR sites with low affinity/low kinase activity EGFR sites and a p53-mediated cell cycle arrest.

The purpose of the present study was to compare the permeaiility and regulation of paracellular transport in human cervical cells with those in epithelial cells of other organs. Cervical cells (ECE16–1, Caski, and HT3) were grown on filters, and trans-epithelial electrical conductance (GT) and the permeability to pyranine (PPyr) were determined. Cervical cultures were characterized by high GT (83–125 mS•cm−2 ) and high PPyr (6.2–18•10−6 •sec−1). The GT was not significantly affected by cell density but was increased by 20% by lowering extracellular calcium to 0.45 mmol/L or less. The high values of GT and PPyr and the regulation by extracellular calcium indicate that all three cervical cell lines have “leaky” tight junctional complexes. Addition of extracellular adenosine triphosphate (ATP) at 50 μmol/L to the cervical cultures evoked a biphasic change in GT- that was unique to the cervical cells: an initial increase, followed by a sustained decrease by 30 /o frorn baseline GT. The decrease oj GT- was associated with a decrease in PPyr by 17%, indicating that ATP had an effect on the tight junctional/paracellular permeability. The ATP effect was reversible either by washing or by chemical hydrolysis with ATPse. 1 he non-cervical cell lines all responded to extracellular ATP with a transient increase in GT, but not with the pronounced decrease. The permeability of the paracellular pathway can be regulated in cervical epithelia by mechanisms that may be different frorn those in epithelial cells frorn other organs.

The P2X(7) receptor is an important regulator of epithelial cell growth. The aim of the present study was to better understand the biological significance of P2X(7) receptor expression in normal and cancer human epithelial tissues. P2X(7) receptor and messenger RNA (mRNA) levels were determined in human tissues containing epithelial dysplastic, pre- or early cancerous, and cancer cells, and the levels were compared to those in the corresponding normal epithelial cells within the same tissue of the same case. P2X(7) receptor levels were determined by quantification of immunoreactivity specific to the functional (full-length) P2X(7) receptor, and P2X(7) mRNA levels were determined by real-time polymerase chain reaction. P2X(7) receptor levels in cancer cells were similar (colon adenocarcinoma) or greater (thyroid papillary carcinoma) than those in the corresponding normal cells. In contrast, in cancer cells of the ectocervix (squamous), endocervix and endometrium (adenocarcinoma), urinary bladder (transitional cell carcinoma), and breast (ductal and lobular adenocarcinomas), P2X(7) receptor levels were lower by about twofold than those in the corresponding normal epithelial cells. Similarly, P2X(7) mRNA levels were lower in uterine, bladder, and breast cancer epithelial tissues by about fourfold than those in the corresponding normal tissues. In addition, P2X(7) receptor levels were decreased already in dysplastic ectocervical cells and pre- or early cancerous endometrial and bladder cells. The data suggest that in epithelia originating from the ectoderm, the uro-genital sinus, and the distal paramesonephric duct, decreased expression of the P2X(7) receptor precedes or coincides with neoplastic changes in those tissues.

Distal (D) segments of the pregnant rat uterine horn express myometrial oxytocin receptors (MORs) earlier than proximal (P) segments (day 18 vs. 20, respectively); the levels in D segments remain higher than in P segments throughout days 21-22 and correlate with the segment-specific myometrial sensitivity to oxytocin. While progesterone (P4) had no effect on MOR levels, RU486 increased (t1/2 6-12 h) MOR levels both in D and P segments, particularly in days 12-17, and the levels in the P segment equaled those in the D segment. Estradiol had no effect on MOR levels in days 20-22; in days 16-19 estradiol increased MOR levels particularly in the P segment, and the levels in the latter were higher than in the D segment. Capillary plasma P4 levels were higher in P vs. in D myometrial segments. These results indicate, in the pregnant rat, a local uterine control of D greater than P MOR expression by P4 withdrawal beginning in day 18. We hypothesize that the D greater than P MOR expression determines a role for oxytocin in initiating myometrial contractions in the D segment, while in active labor another class of agent(s) assume that function in more proximal segments.

The paracellular (intercellular) route is the main transepithelial transport pathway in low-resistance secretory (and absorptive) epithelia, and is composed of the tight junctional resistance (RTJ) and the resistance of the lateral intercellular space (RLIS). Recent studies in human vaginal-cervical epithelia revealed novel mechanisms of regulation of the paracellular resistance (RPCR), including both the RTJ and the RLIS. Steroid hormones and particularly estrogens have been shown to regulate assembled tight junctions as well as the cortical acto-myosin ring, which determine epithelial cell deformability and the RLIS. The knowledge gained from these studies on the molecular mechanisms involved may improve the understanding of dysregulation of water and solutes, and related diseases. The data may be also used in future clinical trials to improve topical drug delivery via the vaginal route in women.

Publisher Summary This chapter describes the epidemiology and pathogenesis of cardiovascular disease (CVD) in women and the risk factors involved, with special emphasis on gender-related differences and relation between estrogen deficiency and heart disease. Ischemic heart disease, commonly referred to as coronary heart disease (CHD), is the single leading cause of CVD deaths in women and in men. CHD in women is predicted to become the pandemic of the 21st century due to escalating increases in CHD risk factors from childhood. The consequences to women could be an earlier onset of CHD, increased prevalence of cardiovascular morbidity and diminished quality of life, decreased life expectancy, and premature death. Active prevention of CHD in women should aim at attenuating the impact of cardiovascular risk factors, beginning as early as childhood. Efforts should include control of eating habits and diet; early detection of plasma lipidemia, diabetes, and hypertension; avoidance of primary and secondary cigarette smoking; and providing education and support for the proper active lifestyle. The role of estrogen replacement in perimenopausal and postmenopausal women for the primary prevention of CVD remains to be determined.

A truncated naturally occurring variant of the human purinergic receptor P2X7 (P2X7-R) was found in human cancer cervical cells. The novel protein consists of 258 amino acids, and compared to the wild-type P2X7-R it lacks the entire intracellular carboxy terminus, the second transmembrane domain, and the distal third of the extracellular loop. The truncated P2X7-R failed to form pores and mediate apoptosis, and it interacted with the wild-type P2X7-R in a manner suggesting auto-hetero-oligomerization. In contrast to cancer cells the novel truncated P2X7-R was expressed relatively little in normal cervical cells. These data raise the possibility that coexpression of the truncated form could block P2X7 mediated apoptosis and promote uncontrolled growth of cells.

The objective of the study was to understand how estrogen modulates the rigidity of the cytoskeleton in epithelial cells. Estrogen depletion decreased, and treatment with 17beta-estradiol increased deformability of cervical-vaginal epithelial cells. Estrogen also induced redistribution of nonmuscle myosin II-B (NMM-II-B); lesser interaction of NMM-II-B with actin; increased phosphorylation of NMM-II-B-heavy chains at threonine and serine residues; and decreased filamentation of NMM-II-B in vitro. The effects of 17beta-estradiol were time and dose related and could be mimicked by diethylstilbestrol. The effects of estrogen were blocked by cotreatment with antisense oligonucleotide for the estrogen receptor-alpha and inhibited by ICI-182,780 and tamoxifen; omission of epithelial growth factor (EGF) from the culture medium; and cotreatments with the EGF receptor inhibitor AG1478, the ERK-MAPK inhibitor PD98059, the casein kinase-II (CK2) inhibitor 5,6-dichloro-1-beta-(D)-ribofuranosylbenzimidazole, the Rho-associated kinase inhibitor Y-27632, and the nonspecific phosphatase inhibitor okadaic acid. Coadministration of 5,6-dichloro-1-beta-(D)-ribofuranosylbenzimidazole plus okadaic acid blocked the 17beta-estradiol effect. H-89 or LY294002 did not significantly affect estrogen effects. Treatment with estrogen increased activation of ERK1/2 and CK2 activity. These data suggest a novel pathway of estrogen regulation of the cytoskeleton in epithelial cells. The effect is mediated by estrogen receptor-alpha and involves in part the EGF-EGF receptor and ERK-MAPK cascades as proximal signaling networks and the CK2 and Rho-associated kinase-regulated myosin heavy chain phosphatase as terminal effectors. Augmented phosphorylation of NMM-II-B can block filamentation and induce disassociation of the myosin from the cortical actin, and disruption of the actomyosin ring can increase cell deformability. This mechanism can explain estrogen regulation of paracellular permeability in cervical-vaginal epithelia in vivo.

A truncated naturally occurring variant of the human receptor P2X7 was identified in cancer cervical cells. The novel protein (P2X7-j), a polypeptide of 258 amino acids, lacks the entire intracellular carboxyl terminus, the second transmembrane domain, and the distal third of the extracellular loop of the full-length P2X7 receptor. The P2X7-j was expressed in the plasma membrane; it showed diminished ligand-binding and channel function capacities and failed to form pores and mediate apoptosis in response to treatment with the P2X7 receptor agonist benzoyl-ATP. The P2X7-j interacted with the full-length P2X7 in a manner suggesting hetero-oligomerization and blocked the P2X7-mediated actions. Interestingly, P2X7-j immunoreactivity and mRNA expression were similar in lysates of human cancer and normal cervical tissues, but full-length P2X7 immunoreactivity and mRNA expression were higher in normal than in cancer tissues, and cancer tissues lacked 205-kDa P2X7 immunoreactivity suggesting lack of P2X7 homo(tri)-oligomerization. These results identify a novel P2X7 variant with apoptosis-inhibitory actions, and demonstrate a distinct regulatory property for a truncated variant to antagonize its full-length counterpart through hetero-oligomerization. This may represent a general paradigm for regulation of a protein function by its variant.

The main function of the acidic environment of the vaginal lumen is to control infections of the lower genital tract. The vaginal acid-base environment is tightly regulated, and during premenopausal years vaginal-fluid pH ranges from 4.5 to 5.5 with mild alkalinization to about 6.0 before ovulation. Despite its important role, relatively little is known about mechanisms that regulate vaginal-fluid pH in women. Until recently, it was believed that the acidic milieu is determined by cohabituating Doderlein lactobacilli.1 We recently presented data that refute the Doderlein theory by showing that human normal vaginal-ectocervical epithelial cells grown on filters under sterile conditions acidify their luminal solution.2 The effect was estrogen-dependent, and could be blocked by low micromolar concentrations of bafilomycin-A1, indicating active proton (H+) secretion by V-type H+-ATPase3 that is located in the apical membrane (Fig. 1). In this regard, vaginal epithelial cells resemble other H+ secreting cells that regulate acid secretion into the lumen.4-9 FIG. 1 Hypothesis of the present study. Vaginal-ectocervical epithelial cells acidify the lumen by active extrusion of protons via proton transport mechanisms located in the apical cell membrane. Tight junctions (filled horizontal bars) separate the plasma membranes ... After menopause, vaginal-fluid pH increases to 6.5 to 7.0.10 Elevated vaginal-fluid pH has been associated with urinary tract infections,11 vaginal atrophy,12 and dyspareunia.13 Treatment with estrogen can acidify the luminal vaginal pH to 5.5 to 6.5,10 suggesting that alkalinization of the vaginal-fluid pH in postmenopausal women is the result of hypoestrogenism. To test this hypothesis more directly, we used our novel culture system of filter generated vaginal-ectocervical cultured epithelia to determine effects of estrogen on luminal proton secretion in cells of postmenopausal women. The results indicate that treatment with estrogen increases active proton secretion by cells of postmenopausal women. However, luminal pH remained elevated compared with that of cells of premenopausal women, suggesting that in addition to hypoestrogenism, other factors of the aging process affect the capacity of vaginal-ectocervical cells to secrete acid.

Treatment of human cervical epithelial CaSki cells with ATP or with the diacylglyceride sn-1,2-dioctanoyl diglyceride (diC8) induced a staurosporine-sensitive transient increase, followed by a late decrease, in tight-junctional resistance (RTJ). CaSki cells express two immunoreactive forms of occludin, 65 and 50 kDa. Treatments with ATP and diC8 decreased the density of the 65-kDa form and increased the density of the 50-kDa form. ATP also decreased threonine phosphorylation of the 65-kDa form and increased threonine phosphorylation of the 50-kDa form and tyrosine phosphorylation of the 65- and 50-kDa forms. Staurosporine decreased acutely threonine and tyrosine phosphorylation of the two isoforms and in cells pretreated with staurosporine ATP increased acutely the density of the 65-kDa form and threonine phosphorylation of the 65-kDa form. Treatment with N-acetyl-leucinyl-leucinyl-norleucinal increased the densities of the 65- and 50-kDa forms. Pretreatment with N-acetyl-leucinyl-leucinyl-norleucinal attenuated the late decreases in RTJ induced by ATP and diC8 and the decrease in the 65-kDa and increase in the 50-kDa forms induced by ATP. Correlation analyses showed that high levels of RTJ correlated with the 65-kDa form, whereas low levels of RTJ correlated negatively with the 65-kDa form and positively with the 50-kDa form. The results suggest that in CaSki cells 1) occludin determines gating of the tight junctions, 2) changes in occludin phosphorylation status and composition regulate the RTJ, 3) protein kinase-C-mediated, threonine dephosphorylation of the 65-kDa occludin form increases the resistance of assembled tight junctions, 4) the early stage of tight junction disassembly involves calpain-mediated breakdown of occludin 65-kDa form to the 50-kDa form, and 5) increased levels of the 50-kDa form interfere with occludin gating of the tight junctions.

Estrogen blocks apoptosis of human ectocervical epithelial cells by modulating P2X7/Ca2+ influx. The effect involves decreased Ca2+-influx and cytosolic-calcium-increase via ATP-activated P2X7 pores. This mechanism may have physiological significance in the human cervix, in-vivo, and the results suggest a physiological role for estrogen in the cervix as an anti-apoptotic factor.

Estrogen blocks apoptosis of human ectocervical epithelial cells by modulating P2X7/Ca2+ influx. The effect involves decreased Ca2+-influx and cytosolic-calcium-increase via ATP-activated P2X7 pores. This mechanism may have physiological significance in the human cervix, in-vivo, and the results suggest a physiological role for estrogen in the cervix as an anti-apoptotic factor.

The objective of this study was to assess estrogen-dependent cellular mechanisms that could contribute to the acid pH of the vaginal lumen. Cultures of normal human cervical-vaginal epithelial (hECE) cells and endocervical cells were grown on filters, and acidification of the extracellular solutions on the luminal (L-pHo) and contraluminal (CL-pHo) sides was measured. The hECE cells and endocervical cells decreased CL-pHo from 7.40 to 7.25 within 20–30 min of incubation in basic salt solution. Endocervical cells also produced a similar decrease in L-pHo. In contrast, hECE cells acidified L-pHo down to pH 7.05 when grown as monoculture and down to pH 6.05 when grown in coculture with human cervical fibroblasts. This enhanced acid secretion into the luminal compartment was estrogen dependent because removal of endogenous steroid hormones attenuated the effect, whereas treatment with 17β-estradiol restored it. The 17β-estradiol effect was dose dependent (EC50 0.5 nm) and could be mimicked by diethylstilbestrol and in part by estrone and tamoxifen. Preincubation with ICI-182780, but not with progesterone, blocked the estrogen effect. Preincubation of cells with the V-ATPase blocker bafilomycin A1, when administered to the luminal solution, attenuated the baseline and estrogen-dependent acid secretion into the luminal solution. Treatment with EGTA, to abrogate the tight junctional resistance, blocked the decrease in L-pHo and stimulated a decrease in CL-pHo, indicating that the tight junctions are necessary for maintaining luminal acidification. We conclude that vaginal-ectocervical cells acidify the luminal canal by a mechanism of active proton secretion, driven in part by V-H+-ATPase located in the apical plasma membrane and that the baseline active net proton secretion occurs constitutively throughout life and that this acidification is up-regulated by estrogen.

The present study explored the effects of estrogen on transcervical tight-junctional resistance (R(TJ)) and the mechanisms involved. Treatment of cultured human cervical epithelial cells with 17beta-estradiol decreased in a time- and dose-related manner the R(TJ). Estrogen had no significant effect on the expression of E-Cadherin, zonula-occluden-1, or Claudin-4. In contrast, 17beta-estradiol modulated expression of the transmembrane tight-junctional protein occludin: at low concentrations (1 and 10 nm) estradiol increased the density of occludin 65-kDa form but at the higher concentration of 100 nm estradiol induced only a mild 2-fold increase in the density of this form. Estradiol also increased the expression of occludin 50-kDa form in a dose-related manner. The R(TJ) and occludin effects of estradiol were reversible and could be blocked by tamoxifen but not progesterone. The present results rule out estrogen modulation of occludin transcription. In contrast, the results suggest that the occludin effects of estrogen involve posttranslational up-regulation of occludin turnover, including synthesis and degradation. The effects of estrogen on occludin expression were compared with those of proteinase-K, plasmin, and matrix-metaloproteinase-2 (all added extracellularly). The three proteinases abrogated irreversibly the R(TJ) and induced expression de novo of occludin low-molecular-weight forms. The latter, however, differed from the effect of estrogen, which generated only a single 50-kDa form. Collectively, the present data suggest that the occludin 50-kDa form is an estrogen-specific-induced occludin isoform and that the mechanism of estrogen-abrogation of transcervical R(TJ) involves occludin modulation.

Normal human ectocervical epithelial (hECE) cells undergo apoptosis in culture. Baseline apoptosis could be increased by shifting cells to serum-free medium and blocked by lowering extracellular calcium. Treatment with the ATPase apyrase attenuated baseline apoptosis, suggesting that extracellular ATP and purinergic mechanisms control the apoptosis. Treatment with ATP and the P2X7receptor analog 2′-3′- O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) increased apoptosis significantly, in a time- and dose-related manner. The threshold of ATP effect was 0.5 μM in hECE cells and ∼1 μM in CaSki cancer cells. The apoptotic effect of BzATP was additive in part to that of tumor necrosis factor (TNF)-α, and it could be attenuated by lowering extracellular calcium and by treatment with the caspase-9 inhibitor Leu-Glu-His-Asp- O-methyl-fluoromethylketone (LEHD-FMK). Treatment with BzATP activated caspase-9, and, in contrast to TNF-α, it had only a mild effect on caspase-8. Both BzATP and TNF-α activated caspase-3, suggesting that BzATP activates predominantly the mitochondrial apoptotic pathway. Both hECE and CaSki cells secrete ATP into the extracellular fluid, and mean ATP activity in conditioned medium was ∼0.5 μM, which is in the range of values that suffice to activate the P2X7receptor. On the basis of these findings we propose a novel autocrine-paracrine mechanism of cervical cell apoptosis that operates by P2X7receptor control of cytosolic calcium and utilizes the mitochondrial apoptotic pathway.