Apically sorted P2X7 receptors mediate purinergic-induced pore formation preferentially in apical domains of the plasma membrane.

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.

Endogenously expressed truncated P2X7 receptor lacking the C-terminus is preferentially upregulated in epithelial cancer cells and fails to mediate ligand-induced pore formation and apoptosis.

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.

Vaginal-cervical epithelial permeability decreases after menopause.

OBJECTIVE: To determine the effects of menopause (aging and E) on vaginal-cervical epithelial paracellular permeability. DESIGN: Experimental, basic clinical research. SETTING: Academic research environment. PATIENT(S): Premenopausal, perimenopausal, and postmenopausal women, aged 35-65 years. INTERVENTION(S): Primary to tertiary cultures of normal human ectocervical epithelial cells on filters. Cells were outgrown from surgically discarded ectocervical minces. MAIN OUTCOME MEASURE(S): Changes in paracellular permeability were determined as changes in transepithelial electrical conductance and pyranine permeability. RESULT(S): [1] Levels of transepithelial electrical conductance and pyranine permeability decreased as women's age advanced. [2] Removal of E from the culture medium decreased paracellular permeability. Treatment of cells in vitro with 10 nmol/L 17beta-E2 increased transepithelial electrical conductance and pyranine permeability, but the effects were additive to the age-related decrease in permeability. [3] Coadministration of 100 nmol/L tamoxifen blocked the E increase in paracellular permeability in cells of both premenopausal and postmenopausal women. CONCLUSION(S): [1] Aging and E deficiency decrease independently vaginal-cervical epithelial paracellular permeability. [2] The E increase in vaginal-cervical epithelial paracellular permeability in cells of postmenopausal women is mediated by the E receptor. [3] The E increase in vaginal-cervical epithelial paracellular permeability in cells of postmenopausal women is masked by age-related increase in the tight junctional resistance, leading to overall decrease in paracellular permeability.

Estrogen biphasic regulation of paracellular permeability of cultured human vaginal-cervical epithelia.

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.

cGMP-dependent ADP depolymerization of actin mediates estrogen increase in cervical epithelial permeability.

Estrogen increases secretion of cervical mucus in women, and the effect depends on fragmentation of the cytoskeleton. The objective of the present study was to understand the molecular mechanism of estrogen action. Treatment of human cervical epithelial cells with 17beta-estradiol, sodium nitroprusside (SNP), or 8-bromoguanosine 3', 5'-cyclic monophosphate (8-Br-cGMP) increased cellular monomeric G-actin and decreased polymerized F-actin. The effects of estradiol were blocked by tamoxifen, by the guanylate cyclase inhibitor LY-83583, and by the cGMP-dependent protein kinase inhibitor KT-5823. The effects of SNP were blocked by LY-83583 and KT-5823, while the effects of 8-Br-cGMP were blocked only by KT-5823. Treatment with phalloidin decreased paracellular permeability and G-actin. Treatment with 17beta-estradiol, SNP, or 8-Br-cGMP attenuated SNP-induced phosphorylation of [(32)P]adenylate NAD in vitro: tamoxifen blocked the effect of estrogen; LY-83583 blocked the effect of SNP but not that of 8-Br-cGMP, while KT-5823 blocked effects of both SNP and 8-Br-cGMP. These results indicate that estrogen, nitric oxide (NO), and cGMP stimulate actin depolymerization. A possible mechanism is NO-induced, cGMP-dependent protein kinase augmentation of ADP-ribosylation of monomeric actin.

Calcium regulates estrogen increase in permeability of cultured CaSki epithelium by eNOS-dependent mechanism.

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-[(3)H]arginine to L-[(3)H]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.

Effects of menopause and estrogen on cervical epithelial permeability.

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).

NO increases permeability of cultured human cervical epithelia by cGMP-mediated increase in G-actin.

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. N(G)-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 Ca(2+)-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.

Role of nitric oxide and cyclic guanosine 3',5'-monophosphate in the estrogen regulation of cervical epithelial permeability.

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.

Involvement of estrogen receptors alpha and beta in the regulation of cervical permeability.

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.

Beneficial effects of estrogen on cardiac stunning in female rabbits.

OBJECTIVE: To determine the effect of estrogen treatment on the contractile, relaxation, and chronotropic responses of hearts of female rabbits to cessation of perfusion. METHODS: Adult female rabbits were treated either with estradiol or with the vehicle (control). The hearts were then isolated and perfused at constant pressure by the Langendorff technique. A saline-filled balloon connected to a pressure transducer was inserted in the left ventricle in order to assess the mechanical function of the isolated heart. Cardiac stunning was induced by halting the perfusion of the coronary vasculature for four successive periods of 1, 3, 5, and 5 minutes, followed by reperfusion between nonperfusion periods. Changes in cardiac function induced by the cessation of perfusion were assessed by monitoring the changes in heart rate and in left ventricular pressure ([dP/dt]max as an index of ventricular contractility and [dP/dt]min as an index of ventricular relaxation). Changes in coronary flow were determined at baseline and following reperfusion. RESULTS: Halting coronary perfusion decreased (dP/dt)max, and (dP/dt)min and slowed left ventricular contractions both in control and in estrogen-treated hearts. The depressant effects of flow cessation on left ventricular (dP/dt)max and (dP/dt)min were smaller in estrogen-treated hearts than in control hearts. Treatment with estrogen had no effect on the changes on the heart rate in responses to cessation of flow and to reperfusion. Treatment with estrogen increased coronary flow by 40%. Coronary reperfusion increased coronary flow transiently, but the effects did not differ significantly between control and estrogen-treated hearts. CONCLUSION: Short-term treatment of adult female rabbits with doses of estrogen that are physiologic for the rabbit exerts a protective effect on cardiac contractility from repetitive periods without perfusion.

Estrogen modulates paracellular permeability of human endothelial cells by eNOS- and iNOS-related mechanisms.

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 (RLIS) and in the resistance of the tight junctions (RTJ); 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 RLIS and RTJ. 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.

Purinergic receptor-induced changes in paracellular resistance across cultures of human cervical cells are mediated by two distinct cytosolic calcium-related mechanisms.

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.

A novel fluorescence chamber for the determination of volume changes in human CaSki cell cultures attached on filters.

The objective of the study was to test the hypothesis that, in the cultured human cervical epithelium, CaSki, the effect of calcium mobilizing agents on transepithelial electrical conductance (GTE), is the result of cell volume decrease. CaSki cells attached on filters were loaded with fura-2, and measurements of fluorescence at the isosbestic wavelength 360 nm (excitation/emission [F360/510]) were made in a newly designed fluorescence chamber; this design allowed us also to determine changes in cytosolic calcium ([Ca2+]i). The experimental conditions were similar to those used to measure changes in paracellular permeability in the Ussing chamber, and they enabled us to compare the time-course of changes in [Ca2+]i, in F360/510, and in GTE. Hypertonicity increased, and hypotonicity decreased F360/510 and GTE, without having an effect on [Ca2+]i, and the changes in F360/510 and in GTE correlated linearly. Metabolism, bleaching, and extrusion of intracellular fura-2 were minimal, indicating that the changes in F360/510 reflect changes in dye concentration. Hypertonicity decreased, and hypotonicity increased the size of dispersed CaSki cells, suggesting that osmolarity-induced changes in F360/510 reflect changes in size of the attached cells. Ionomycin increased [Ca2+]i, F360/510, and GTE, but the increases in [Ca2+]i preceded those in F360/510 and GTE. The calcium chelator BAPTA blocked the ionomycin-induced increase in [Ca2+]i, F360/510, and in GTE. Preincubation with 4-acetamido-4'isothiocyanatostilbene-2,2'disulfonic acid (SITS) augmented the ionomycin-induced increase in [Ca2+]i, but blocked the increases in F360/510 and in GTE. Pretreatment of cells with hypertonic solution abrogated the increases in F360/510 and in GTE in response to ionomycin, but had little effect on the ionomycin-induced increase in [Ca2+]i. On the basis of these results we suggest that the ionomycin-induced increase in GTE is mediated by [Ca2+]i-dependent chloride secretion and osmotic water loss.

Effects of estrogen on tight junctional resistance in cultured human umbilical vein endothelial cells.

OBJECTIVE: To study the effects of estrogen on transendothelial paracellular permeability in women. METHODS: Human umbilical vein endothelial cells (HUVEC) obtained from women were grown on filters. The paracellular permeability characteristics were determined in terms of changes in the permeability to the polar acid pyranine (Ppyr) and as changes in the transendothelial electrical resistance (RTE). Tight junctional resistance characteristics were assayed by lowering luminal NaCl and measuring the dilution potential, and were expressed as the ratio of monoion mobility uCl/uNa (cation selectivity). RESULTS: Low extracellular calcium and hyperosmolarity increased Ppyr and decreased RTE. The former but not the latter condition abolished the endothelium-specific cation selectivity. Treatment with 10 nM of estradiol-17 beta had no effect on RTE, but it increased the cation selectivity. The effect of estradiol required 1-6 hours' incubation with the hormone; it was dose dependent and saturable, with a median effective concentration of estradiol of 1 nM. Diethylstilbestrol, but not estriol, could mimic the effect of estradiol, and the estrogen receptor antagonist ICI-182, 780 blocked it. CONCLUSION: Cultured HUVEC cells form patent tight junctions. Estrogens increase the cation selectivity across HUVEC cultures. The effect of estrogen may be mediated by an estrogen receptor. These effects may be important for vasculoprotection in cases of sudden changes in ions levels across the capillary wall, such as ischemia or reperfusion.

Regulation by retinoids of P2Y2 nucleotide receptor mRNA in human uterine cervical cells.

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 P2Y2 receptor, 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 the cultured human cervical epithelium by modulating cell deformability.

Estrogens increase secretion of cervical mucus in females. The objective of this research was to study the mechanisms of estrogen action. The experimental models were human CaSki (endocervical) and hECE (ectocervical) epithelial cells cultured on filters. Incubation in steroid-free medium increased transepithelial electrical resistance (RTE) and decreased epithelial permeability to the cell-impermeant acid pyranine. Estrogen treatment reversed the effects, indicating estrogen decreases epithelial paracellular resistance. The estrogen effect was time and dose related (EC50 approximately 1 nM) and specific (estradiol = diethylstilbestrol > estrone, estriol; no effect by progesterone, testosterone, or cortisol) and was blocked by progesterone, tamoxifen, and ICI-182780 (an estrogen receptor antagonist). Estrogen treatment did not modulate dilution potential or changes in RTE in response to diC8 or to low extracellular Ca2+ (modulators of tight junctional resistance). In contrast, estrogen augmented decreases in RTE in response to hydrostatic and hypertonic gradients [modulators of resistance of lateral intercellular space (RLIS)], suggesting estrogen decreases RLIS. Estrogen decreased cervical cell size, shortened response time relative to changes in cell size after hypertonic challenge, and augmented the decrease in cell size in response to hypertonic and hydrostatic gradients. Lowering luminal NaCl had no significant effect on RTE, and the Cl- channel blocker diphenylamine-2-carboxylate attenuated the hypertonicity-induced decrease in cell size to the same degree in control and estrogen-treated cells, suggesting estrogen effects on permeability and cell size are not mediated by modulating Na+ or Cl- transport. In contrast, estrogen increased cellular G-actin levels, suggesting estrogens shift actin steady-state toward G-actin and the cervical cell cytoskeleton toward a more flexible structure. We suggest that the mechanism by which estrogens decrease RLIS and increase permeability is by fragmenting the cytoskeleton and facilitating deformability and decreases in cervical cell size.

Modulation of coronary vascular resistance in female rabbits by estrogen and progesterone.

OBJECTIVE: To study the roles of estradiol and various progestins on the regulation of coronary flow in female rabbits. METHODS: Ovariectomized adult female rabbits were treated with estradiol, with progesterone (or one of the following synthetic progestins: megace, norethindrone, or medroxyprogesterone acetate), or with both an estradiol and a progestin. Hearts were isolated and perfused at constant pressure by a modified Langendorff technique. Changes in coronary flow were determined at baseline and in response to direct infusion into the coronary circulation of NG-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide (NO) synthase. RESULTS: Coronary flow rates were 40-50% greater in hearts of animals treated with estradiol than in control hearts of animals not treated with the hormone. Treatment of the animals with progestin alone had little effect on coronary flow. However, when administered with estradiol, it abrogated the estradiol-related increase in coronary flow. The increments in coronary flow evoked by estradiol were virtually abolished by L-NNA, an inhibitor of NO synthase. In hearts of animals treated with estradiol plus progesterone, L-NNA had no additional inhibitory effect on coronary flow to that of progesterone. CONCLUSION: Estradiol decreases coronary vascular resistance (CVR) and hence increases coronary flow. Progestins attenuate this effect of estradiol.