In vitro models of mucosal HIV transmission.
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Animals↗
Biomedical subjects
G I Gorodeski
Publications and source records attributed to G I Gorodeski.
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.
Adult↗
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.
Actins↗
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.
Alkaloids↗
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).
Adult↗
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.
Arylsulfonates↗
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.
Adenosine Triphosphate↗
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.
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
Seminal fluid factor increases the resistance of the tight junctional complex of cultured human cervical epithelium CaSki cells.
OBJECTIVE: To determine the effects of human seminal fluid on cervical paracellular resistance. DESIGN: Experimental study. SETTING: Healthy volunteers in an academic research environment; cultures of human CaSki cells on filters, with phenotypic characteristics of the endocervix. PATIENT(S): Healthy men donating sperm to a sperm bank. INTERVENTION(S): Seminal fluid was obtained as the discarded fluid from ejaculates. MAIN OUTCOME MEASURE(S): Changes in transepithelial electrical resistance across CaSki cells on filters were determined in an Ussing chamber from successive measurements of the short-circuit current and the transepithelial potential difference. Changes in the dilution potential (and hence in the ratio of Cl- to Na+ mobilities) were determined after lowering the NaCl concentration in the luminal solution. RESULT(S): Seminal fluid increased transepithelial electrical resistance acutely (t1/2, 2 minutes), reversibly, and in a dose-related manner (ED50, 1%). The effect of seminal fluid was abolished when the extracellular calcium level was lowered, and the increase in transepithelial electrical resistance correlated with a decrease in the ratio Cl- to Na+ mobilities, indicating an increase in the resistance of the tight junctional complex. The increase in transepithelial electrical resistance in response to seminal fluid was nonadditive to that of sn-1,2-dioctanoyl diglyceride (a stable diacylglyceride and activator of protein kinase C), and it was abolished by prolonged preincubation with the phorbol ester phorbol 12-myristate 13-acetate (to downregulate protein kinase C) or with staurosporin (to inhibit protein kinase C), suggesting that seminal fluid acts through a protein kinase C-dependent mechanism. Slower (t1/2, 3.3 minutes) increases in transepithelial electrical resistance occurred when seminal fluid was added only to the luminal or the subluminal solution. Treatment with pertussis toxin, adenosine triphosphatase, or trypsin had no effect on the changes in transepithelial electrical resistance. Seminal fluid increased cytosolic calcium, but changes in cytosolic calcium are not important for the increases in transepithelial electrical resistance, suggesting that the effect of seminal fluid is not receptor-mediated. Preliminary studies indicate that the factor(s) in seminal fluid that increases transepithelial electrical resistance is a labile, low molecular weight (< 10 kd) lipid. CONCLUSION(S): Seminal fluid may regulate cervical mucus production in vivo by modulating endocervical permeability.
Calcium↗
Retinoids modulate P2U purinergic receptor-mediated changes in transcervical paracellular permeability.
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 concentrations (K 1/2) = 0.1 microM] were required for restoring the phase I response than were required for reversing the phase II response (K 1/2 = 1 microM). 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 II response 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.
Adenosine Triphosphate↗
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.
3T3 Cells↗
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.
Actins↗
Extracellular Ca2+ directly regulates tight junctional permeability in the human cervical cell line CaSki.
Lowering extracellular calcium concentration ([Ca2+]o) increases acutely and reversibly the transepithelial electrical conductance (G(TE)) and the epithelial permeability to pyranine (Ppyr) across CaSki cultures. Effects were already observed after lowering calcium from 1.2 to 1.0 mM and were maximal at 0.1 mM. The dose-response curves were sigmoidal (calcium concentration that produces half-maximal effect = 0.3 mM), and the time courses indicated simple exponential trends (time constants of 4-5 min). The effect of calcium was not mediated by mobilization of cytosolic calcium or altering calcium influx, and manganese was found to be a partial agonist to [Ca2+]o. The effects of [Ca2+]o, on permeability were additive to those of hypertonic conditions, indicating that calcium modulates junctional permeability. The experimental data were fitted to theoretical models that relate changes in G(TE) to the probability of assembled/disassembled tight junctions. The results suggest that calcium interacts directly and cooperatively at extracellular sites with junctional elements that are arranged in parallel, and it shifts the probability state of the junctions from "open" to "closed" state. Changes in extracellular calcium may affect the permeability of tight junctions of the cervical epithelium and may play a role in regulating production of cervical mucus.
Calcium↗
Extracellular ATP regulates transcervical permeability by modulating two distinct paracellular pathways.
Extracellular ATP stimulates a biphasic change in transepithelial electrical resistance (RTE) 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 (RLIS) and the tight junctional resistance (RTJ) to the changes in RTE. 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 Ca2+ 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 RLIS 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 RTJ in day 2-6 cultures; the phase I decrease of 50% in RLIS 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 RTE. We conclude that ATP regulation of RLIS and RTJ may be important mechanisms of modulating cervical mucus production in vivo.
Adenosine Triphosphate↗
Retinoids regulate tight junctional resistance of cultured human cervical cells.
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.
Cell Line↗
Nucleotide receptor-mediated decrease of tight-junctional permeability in cultured human cervical epithelium.
Extracellular ATP changes the transepithelial electrical conductance (GT) across cultures of human cervical cells acutely, in a biphasic manner that is characterized by a rapid increase (phase I) followed by a sustained decrease in GT (phase II). We tested the hypothesis that the phase II response is mediated by decreases in the permeability of tight junctions. We studied the effect of ATP on the relative mobilities of Cl- vs. Na+ (uCl/uNa) as calculated from changes in the dilution potential (Vdil). Vdil was induced by lowering NaCl from 130 to 10 mM in either the luminal or subluminal solutions bathing filters containing cells. uCl/uNa was 1.27 across cervical cultures and 1.34 across blank filters, compared with a level of 1.52 in free solution. Increases in GT induced by transepithelial hydrostatic or hypertonic gradients (which increase permeability of lateral intercellular space) had no effect on uCl/uNa. Increases in GT induced by lowering extracellular Ca2+ to < 0.1 mM increased uCl/uNa to levels obtained in blank filters, indicating abrogation of tight-junctional resistance. Phase I response and ionomycin (which produces a sustained phase I-like increase in GT) had no effect on uCl/uNa. The phase II response, however, decreased uCl/uNa from 1.27 to 1.24, and the effect could be abrogated by lowering extracellular Ca2+. These results indicate that phase II decreases in GT across cultured human cervical epithelium are mediated by acute decreases in tight-junctional permeability.
Adenosine Triphosphate↗
Biphasic regulation of paracellular permeability in human cervical cells by two distinct nucleotide receptors.
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.
Adenosine Triphosphate↗
Osmolar changes regulate the paracellular permeability of cultured human cervical epithelium.
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.
Adenosine Triphosphate↗