Estradiol had a biphasic effect on permeability across cultures of human umbilical vein endothelial cells (HUVEC): at nanomolar concentrations it decreased the HUVEC culture permeability, but at micromolar concentrations it increased the permeability. The objective of the present study was to test the hypothesis that the changes in permeability were mediated by nitric oxide (NO)-related mechanisms. The results revealed dual modulation of endothelial paracellular permeability by estrogen. 1) An endothelial NO synthase (eNOS)-, NO-, and cGMP-related, Ca2+-dependent decrease in permeability was activated by nanomolar concentrations of estradiol, resulting in enhanced Cl−influx, increased cell size, and increases in the resistance of the lateral intercellular space ( R LIS) and in the resistance of the tight junctions ( R TJ); these effects appeared to be limited by the ability of cells to generate cGMP in response to NO. 2) An inducible NO synthase (iNOS)- and NO-related, Ca2+-independent increase in permeability was activated by micromolar concentrations of estradiol, resulting in enhanced Cl−efflux, decreased cell size, and decreased R LIS and R TJ. We conclude that the net effect on transendothelial permeability across HUVEC depends on the relative contributions of each of these two systems to the total paracellular resistance.
Extracellular ATP stimulates acute changes in paracellular permeability across cultures of human uterine cervical epithelial cells [G. I. Gorodeski, D. E. Peterson, B. J. De Santis, and U. Hopfer. Am. J. Physiol. 270 ( Cell Physiol. 39): C1715–C1725, 1996]. In this paper, we characterize mRNA for a P2Y2 nucleotide receptor in human cervical cells. Using oligonucleotide primers based on the sequence of human airway epithelium P2Y2receptor, a single 632-bp cDNA band was identified in RT-PCR experiments in extracts of human endocervical and ectocervical tissues and in lysates of human cervical CaSki cells, but not in 3T3 fibroblasts. The nucleotide sequence was homologous to the corresponding human airway epithelium P2Y2 receptor. Northern blot analyses revealed hybridization of the P2Y2 receptor probe to a 2.0-kb mRNA fragment, as well as to 2.2-, 3.0-, and 4.6-kb species, indicating that human cervical cells express P2Y2 receptor mRNA. Incubation of CaSki cells in retinoid-free medium abolished the ATP-induced changes in permeability and decreased the expression of the P2Y2 receptor mRNA; treatment with retinoids restored the responses to ATP and upregulated the P2Y2 receptor mRNA, suggesting that the receptor mediates ATP-related changes in permeability. Treatment with actinomycin D decreased the expression of the P2Y2 receptor RNA, but the ratio density of the receptor RNA relative to glyceraldehyde-3-phosphate dehydrogenase RNA remained unchanged, suggesting that retinoids upregulate transcription of the receptor mRNA. We conclude that retinoid-dependent modulation of the P2Y2 receptor expression, and hence of the responses to ATP, may be an important mechanism for the regulation of secretion of cervical mucus in vivo.
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.
The objective of the study was to determine the effect of retinoids on paracellular resistance across the cervical epithelium and the mechanisms involved. The experimental model was cultures of human CaSki cells on filters, which retain phenotypic characteristics of the endocervical epithelium. End points for paracellular resistance were measurements of transepithelial electrical resistance and fluxes of pyranine (a trisulfonic acid that traverses the epithelium via the intercellular space). Paracellular resistance was significantly increased in cells grown in retinoid-free medium; the effect could be blocked and reversed with all- trans-retinoic acid (tRA) and with agonists of RAR and RXR receptors but only partially with retinol. The effect of tRA was dose dependent and saturable, with a 50% effective concentration of 0.8 nM. The increases in paracellular resistance induced by vitamin A deficiency required longer incubation in retinoid-free medium than decreases in resistance induced by retinoic acid. tRA had only a minimal effect on paracellular resistance in cells maintained in regular medium. Retinoid-free medium increased and tRA decreased the relative cation mobility across CaSki cultures. Also the effects of tRA were nonadditive to those of cytochalasin D (which decreases tight junctional resistance) and additive to those of ionomycin (which decreases the resistance of the lateral intercellular space), suggesting that tRA modulates tight junctional resistance. It is concluded that vitamin A determines the degree of paracellular resistance across cervical cells by a mechanism that involves modulation of tight junctional resistance.
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 ( K ½) = 0.1 μM] were required for restoring the phase I response than were required for reversing the phase II response ( K ½ = 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.
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