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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.

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.

The effects of extracellular ATP (ATPe) were characterized in human cervical cells grown on filters. ATPe changed the transepithelial conductance (GT) in a biphasic manner with an initial acute increase of 13 +/- 3% (phase I), lasting approximately 2 min, followed by a sustained decrease of 49 +/- 17% (phase II). ATPe also effected decreases of permeabilities to pyranine and to sucrose, which correspond to the phase II decrease in GT. Both phase I and II effects appear to be mediated by apical nucleotide receptors. However, the phase I effect differed from the phase II effect as follows: 1) a higher 50% effective concentration for ATPe, 22 vs. 3 microM; 2) different nucleotide specificity; 3) lack of influence of pretreatment with pertussis toxin; 4) independence from time in culture after seeding; and 5) involvement of cytosolic calcium, rather than diacylglycerol, as intracellular messenger. These differences suggest the presence of two different types of nucleotide receptors that use different intracellular signaling systems and have opposite effects on the paracellular permeability and electrical conductance. These results suggest that ATPe could regulate cervical mucus production in vivo by regulating the paracellular permeability. Depending on the specific receptors present, ATPe could either increase or decrease mucus production.

Objective: The purpose of the present study was to compare the permeability and regulation of paracellular transport in human cervical cells with those in epithelial cells of other organs., Methods: Cervical cells (ECE16-1, Caski, and HT3) were grown on filters, and transepithelial electrical conductance (GT) and the permeability to pyranine (PPyr) were determined., Results: Cervical cultures were characterized by high GT (83-125 mS.cm-2) and high PPyr (6.2-18 x 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 mumol/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% from baseline GT. The decrease of 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 ATPase. The non-cervical cell lines all responded to extracellular ATP with a transient increase in GT, but not with the pronounced decrease., Conclusion: The permeability of the paracellular pathway can be regulated in cervical epithelia by mechanisms that may be different from those in epithelial cells from other organs.

We studied the effect of ATP on transepithelial transport through the paracellular pathway in human cervical cells. Transepithelial conductance and transepithelial permeability (determined from the measurements of unidirectional flux of inert molecules) were measured in Caski cells grown on permeable support. Transepithelial conductance was 55.9 +/- 17.7 mS/cm2 and permeability was 12.5 +/- 2.7 x 10(-6) cm/s for a 0.51-kDa probe. Addition of ATP to the medium decreased acutely and reversibly the conductance and the permeability to probes between 0.18 and 10 kDa by 23-31% in a dose-related fashion; the 50% effective concentration was 1 microM, with a maximal effect at 5-10 microM extracellular ATP. The ATP effect was observed regardless of the pressure gradient across the epithelium. These results indicate that extracellular ATP in micromolar concentrations decreases acutely and reversibly the permeability through the paracellular pathway in cervical cells, possibly by affecting the permeability of the tight junctions and the resistance of the intercellular space. On the basis of these data, we speculate that ATP may play a role in the regulation of solutes and fluid transport across the cervical epithelium in vivo.

The purpose of the present study was to establish culture conditions for human uterine cervical epithelial cells on permeable support and to determine how it affects cervical cell differentiation. Human ectocervical epithelial cells (hECE), HPV-16 immortalized hECE cells (ECE16-1) and Caski cells were grown on collagen-coated filters. Culture conditions, density of cells in culture and expression of epithelial and cervical-cell phenotypic markers were determined and compared in cells grown on filter and on solid support. Compared with the latter, cultures on filter had a higher cell density, hECE cells stratified to 5-12 cell layers compared to 1-3 on solid support, and cells of all three types expressed intercellular tight junctions. The cytokeratin profiles revealed differences between the three cell types as well as differences within the same cell species when grown on filter, compared to solid support. Of particular importance was the finding of a higher expression of K-13 in hECE grown on filter compared to solid support; K-13 is a marker of ectocervical cell differentiation. The cytokeratin profiles of the cultured hECE, ECE16-1 and Caski cells resembled those of ectocervical, squamous metaplastic and endocervical epithelia, respectively. hECE and ECE16-1 expressed involucrin protein, the level of which in both was higher in cells grown on filter compared to solid support. Polarization of the cultures was determined by morphology (stratification of hECE cells, expression of pseudomicrovilli in the apical cell membrane), selective apical vs. basolateral secretion of [35S]methionine- and [35S]cysteine-, [3H]fucose- and [14C]glucosamine-labeled molecules, and positive short-circuit current (Isc) under voltage-clamp conditions. Confluency of the cultures was determined by measuring transepithelial unidirectional fluxes of inert molecules with different molecular weights (MWs) through the paracellular pathway, and by measuring transepithelial conductance. The results indicated transepithelial permeability of 7-22.10(-6) cm.sec-1, which was 5-100 fold smaller compared to blank inserts, with a cut-off MW of 40-70 kDa for hECE and Caski cells. Transepithelial conductance ranged 18.5 to 51.5 mS.cm-2, indicating a leaky but confluent epithelia. Collectively the results indicate the epithelial nature of the cells and their improved differentiation when grown on filter support; hECE is a model for ectocervical epithelium while ECE16-1 and Caski express phenotypic characteristics of squamous metaplastic cervical epithelium and endocervical epithelium respectively.

During the menstrual cycle, the ectocervical epithelium undergoes a cyclic change in differentiation that is correlated with changes in the circulating levels of estrogens and progestins. To better understand the mechanisms underlying this regulation, we have developed an ectocervical epithelial cell (ECE cell) culture system that responds in a physiological manner to stimulation by estrogens and progestins. We have recently proposed that four classes of hormones (retinoids, estrogens, progestins, and glucocorticoids) may play an important role in regulating differentiation. In the present paper we test this hypothesis directly by making detailed dose-response curves. Our results demonstrate that estrogen increases ECE cell differentiation, as measured by release of cornified envelopes (superficial cells), while progesterone decreases envelope production. The effects are dose dependent and within the expected physiological range (0.01-10 nM estrogen; 0.1-100 nM progesterone). Very low concentrations of Ro 13-6298 (0.01-10 nM), a synthetic retinoid, decreased envelope production, while hydrocortisone (0.1-50 nM) increased envelope production and cell growth. Importantly, agents that enhance envelope production are neutralized by agents that reduce envelope formation and vice versa. Based on these findings we conclude 1) that the differentiation-enhancing actions of glucocorticoids and estrogens can be antagonized by either progestins or retinoids, and 2) that glucocorticoids and retinoids are likely to determine the ECE cell differentiation set-point in vivo, with the sex steroids directly modulating the phenotype of the ECE cells around this set-point during the menstrual cycle. Moreover, these results appear to explain some of the clinical descriptions of changes in ectocervical cell morphology resulting from hyper- and hypoestrogenic stimulation.

In the present manuscript we demonstrate that ectocervical epithelial cells (ECE cells) retain a high degree of differentiated function when cultured using feeder layers. We characterize the cultured cells with respect to morphology, expression of cytokeratins, responsiveness to sex steroids, and the presence of estrogen-binding sites. Like ectocervical cells in vivo, the cultured cells display a typical epithelial cell morphology and undergo extensive stratification and envelope (superficial cell) formation. Like the in vivo ectocervical epithelium, the cultured ECE cells express type I cytokeratins K13, K14, K16, K17, and K19 and type II cytokeratins K5 and K6. Under normal culture conditions, however, cytokeratins K1, K2, K4, K11, and K15, which are expressed in vivo, are not expressed. An interesting finding is that ECE cells, in contrast to endocervix and epidermis in vivo and cultured epidermal keratinocytes, express very abundant levels of K13. In fact, K13 appears to be a specific marker of ECE cells in the female reproductive tract. When incubated with 10 nM diethylstilbestrol, ECE cell envelope production increased 3-fold, while incubation with 100 nM progesterone decreased envelope formation 3.4-fold. Simultaneous incubation with progesterone antagonized the diethylstilbestrol stimulation. Thus, in vivo-like sex steroid regulation of ECE cell differentiation is maintained in culture. In addition, the cells possess a high affinity, limited capacity binding site for estradiol that has a Kd of 1.2 +/- 0.1 nM. This system is likely to provide a useful model for the study of sex steroid regulation of normal ectocervical epithelial cell function.

In spite of extensive study of the reproductive tract, little knowledge is available regarding the function of ectocervical epithelial (ECE) cells. In the present study we utilized a feeder layer of 3T3 cells to grow homogeneous cultures of human ectocervical epithelial cells and demonstrated the presence of the cornified envelope precursor, involucrin. Treatment of these cultures with 1 nM Ro 13-6298, a synthetic analogue of trans-retinoic acid, suppresses envelope formation 6-fold with half-maximal suppression at 0.005-0.01 nM. Treatment with 1 microM hydrocortisone elevates envelope production 2.5-fold. Sex steroids also regulate desquamation: 10 nM diethylstilbestrol, a synthetic estrogen, increases envelope levels 2- to 3-fold, while 300 nM progesterone reduces envelope production 2- to 3-fold. In spite of the retinoid-, glucocorticoid- and sex-steroid-stimulated changes in envelope production, the level of the envelope precursor, involucrin, remains constant. Our results suggest: (1) that, in vivo, ectocervical cell squame formation is regulated by the combined direct action of estrogens, progestins, glucocorticoids and retinoids; and (2) that envelope formation is not regulated by changes in the cellular content of the envelope precursor, involucrin. We present a model summarizing the estrogen, progestin, glucocorticoid and retinoid effects on ectocervical epithelial cell function.