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Biology of Reproduction 61, 657-664 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Effect of Inflammatory Mediators on the Electrophysiology of the Human Oviduct1

S.J. Downing2,a, E.L. Chambersa, S.D. Maguinessb, A. Watsonb, and H.J. Leesea

a Department of Biology, University of York, York YO21 5YW, United Kingdom b The Princess Royal Hospital, Hull HU8 9HE, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of histamine and other inflammatory mediators on the electrophysiology and intracellular free calcium ([Ca2+]i) of human oviductal epithelial cells, grown as a polarized layer in primary culture, were studied. Transepithelial potential difference (PD) and short-circuit current (Iscc) were recorded using a modified Ussing chamber. Resistance (R) was calculated from the measurements of PD and Iscc. Basally applied histamine produced transient increases in PD and Iscc with a small decrease in R. The histamine effect was reduced by triprolidine (H1 receptor antagonist) but was unaffected by H2 (ranitidine) or H3 (thioperamide) receptor antagonists. Blockers of Na+, K+, or Na+/K+/2Cl- channels did not affect histamine action. Blockers of Cl-/HCO3- channels or Ca2+ channels reduced the histamine effect. Platelet activating factor (PAF), applied apically, increased PD and Iscc. Histamine produced a transient increase in fluorescence of Fura 2-AM dye, indicating an increase in [Ca2+]i. Triprolidine pretreatment inhibited histamine-stimulated [Ca2+]i increase. Cimetidine, (H2 receptor antagonist), ranitidine, or thioperamide reduced the histamine effect. Histamine increased contractions of both circular and longitudinal smooth muscles in oviduct segments, an effect that was antagonized by triprolidine or thioperamide but not by ranitidine. Histamine's action on Ca2+ and Cl- movements may adversely affect oviductal fluid production and decrease fertility. PAF's effects on Cl- movements may be important for normal embryo transport.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The oviduct is the site of ovum and sperm transport, fertilization, and early embryo development. Tubal fluid, secreted by the epithelial cells lining the inner surface of the oviduct, provides the optimum environment for these processes, but the mechanism and regulation of its secretion are poorly understood [1]. Movement of fluids across epithelia are secondary to the movement of solutes, particularly ions. Using a vascular perfusion technique, Gott et al. [2] and Dickens and Leese [3] found that basal to apical movements of chloride ions were associated with tubal fluid secretion in the rabbit oviduct. These chloride fluxes were sensitive to blockade of Cl-/HCO3- exchange and Na+/K+/2Cl- cotransport [2], which is consistent with the oviduct being a Cl- secretory epithelium as is found in other tissues such as the kidney proximal tubule and the airways. Dickens et al. [4] then grew rabbit oviduct epithelial cells as a polarized layer in primary culture. The cultured cells showed structural polarization with the basal side in contact with the filter and the apical surface covered in microvilli and cilia. Functional polarization of the cells was also demonstrated by asymmetric distribution of glucose disappearance and lactate appearance. Using this technique, it was shown that chloride ion flux in the secretory (basal to apical) direction was greater than the absorptive (apical to basal) direction and that transepithelial electrical potential difference (PD) increased in response to adrenergic agonists. Dickens et al. [5] showed that human tubal epithelial cells could also be grown in a polarized manner. Immunocytochemical analyses demonstrated that a pure population of epithelial cells were isolated and that the cells formed extensive desmosome junctions in culture. We have subsequently shown that the movement of chloride ions is an important factor in the generation of the transepithelial potential difference across these cultured human oviductal epithelial cells and that extracellular ATP is a potent modulator of electrophysiological activity [6].

Tubal fluid production may be perturbed in cases of pelvic inflammatory disease or hydrosalpinx, leading to reduced fertility. It has been suggested that the retrograde spill of hydrosalpingeal fluid may adversely affect endometrial receptivity, may be embryotoxic [7], or may provide a medium for embryo development that lacks essential components [8, 9]. It has also been suggested that constant passage of fluid may cause mechanical interference resulting in failure of implantation [10]. Surgical removal of the hydrosalpinx has been advocated prior to in vitro fertilization, to remove the deleterious effects of inflammatory mediators on the uterine endometrium. This procedure, however, is controversial and has not been found to improve pregnancy rates in all clinics. It is important to determine the effect of inflammatory mediators such as histamine, platelet activating factor (PAF), leukotrienes, and prostaglandins on human tubal function.

Perturbation of tubal function by inflammatory mediators may not be limited to abnormalities in tubal fluid production. Compounds, such as histamine, have been shown to stimulate smooth muscle contraction in a number of tissues [11]. Leukotriene B4 enhances smooth muscle contractions induced by histamine or acetyl choline [12]. PAF has also been shown to induce bronchoconstriction [13], stimulate myometrial contractions [14, 15], and promote synthesis of prostaglandin E2 [16, 17]. It is possible that reduced fertility associated with hydrosalpinx may also be due to abnormal myosalpingeal contractile activity that adversely affects ovum and embryo transport.

PAF may act not only as an inflammatory mediator but also as an embryonic signal that hastens embryo transport down the oviduct to the uterus. PAF has been detected in preimplantation embryos of mouse [18], hamster [19], and bovine [20]; and receptors for PAF have been located in the endosalpinx of the hamster [21]. PAF has been shown to increase intracellular calcium concentrations in cultured bovine oviduct endosalpingeal cells [22] via influx of extracellular calcium. It is likely, therefore, that PAF has significant effects on the electrophysiological responses and intracellular calcium concentrations of epithelial cells of human oviducts.

We have therefore investigated the effects of inflammatory mediators on the electrical properties and intracellular free calcium concentrations in human tubal epithelial cells and on the contractile activity of the myosalpinx.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human oviducts, at various stages of the menstrual cycle, were removed from 40 premenstrual patients attending for hysterectomy at the Princess Royal Hospital, Hull, UK. Permission was granted by the Hull and East Yorkshire Ethics and Clinical Trials Committee and patient's informed consent was always obtained. Oviducts were taken from 9 patients at the proliferative stage of the menstrual cycle and 5 patients at the secretory stage; 5 patients were menstruating at the time of surgery; and 19 patients had irregular menses or longer than four weeks since the last menstrual period. Two patients had received Depo-Provera (Pharmacia and Upjohn, Kalamazoo, MI).

Epithelial Cell Culture

Epithelial cells were isolated according to the method of Dickens et al. [4], which is a modification of methods devised by Glasser et al. [23] and Kimber et al. [24]. The oviducts were washed in Hanks' balanced salt solution without calcium and magnesium (Ca-Mg-free HBSS; Gibco, Life Technologies, Ltd., Paisley, UK) and connective tissue removed. The oviducts were opened longitudinally, chopped into 1-cm pieces, and incubated in Ca-Mg-free HBSS containing 0.5% type I trypsin (Sigma Chemical Co., Poole, UK) and 2.7% (w:v) pancreatin (Gibco) for 1 h at 4°C. This was followed by a further incubation for 1 h at room temperature. The enzyme medium was removed, Ca-Mg-free HBSS added, and the epithelial cells vortexed into suspension. After centrifugation at 250 x g for 5 min, the cells were washed in Ca-Mg-free HBSS and recentrifuged 3 times. After washing, cells were resuspended in prewarmed, pregassed culture medium at a density of 1 x 106 cells/ml. Cell viability, tested by the ability to exclude trypan blue (0.4% w:v), was more than 95%. Culture medium consisted of nutrient mixture F12 (Sigma) plus DMEM (1:1, v:v) (Sigma) containing 0.1% BSA (ICN-Flow, Oxfordshire, UK), 270 U/ml penicillin, 270 µg/ml streptomycin (Sigma), 20 µg/ml fungizone (Gibco), 2.5 mM glutamine (Sigma), 2.5% Nu-serum (ICN-Flow), and 2.5% heat inactivated fetal calf serum (Gibco).

For electrophysiological studies, 250 µl of the cell suspension was placed in the top of Snapwell filters, 1 cm2, 0.4-µm pore size (Corning Costar Corporation, Cambridge, MA) coated with Pronectin F (Protein Polymer Technologies, Inc., San Diego, CA) in multiwell plates, and 4 ml fresh medium was added under the filters. The cells were incubated in a humidified incubator at 37°C, gassed with 5% CO2 in air. The medium was replaced above and below the filters every 48 h. When the cells became confluent, the filters were placed in modified Ussing chambers (World Precision Instruments, Inc., Sarasota, FL), with both surfaces of the cells bathed with normal Krebs Ringer bicarbonate solution and gassed with 95% O2/5% CO2, at 37.5°C. Using pairs of glass KCl electrodes, the cells were alternately clamped electrically at 0 mV for 5 sec and +5 µA for 1 sec. This permitted almost simultaneous recording of potential difference (PD) and short-circuit current (Iscc). Resistance (R) was calculated from the measurements of PD and Iscc. The normal Krebs Ringer bicarbonate solution contained: 118 mM NaCl, 25 mM NaHCO3, 4.74 mM KCl, 1.19 mM MgSO4, 1.17 mM KH2PO4, 1.17 mM CaCl2, 1 mM glucose, gassed with 95% O2/5% CO2. In chloride-free medium, NaCl, KCl, and CaCl2 were replaced with sodium gluconate, potassium gluconate, and calcium gluconate, respectively. Drugs were made up in normal Krebs Ringer bicarbonate, or in chloride-free medium, and added in 40-µl volumes to either the basal or apical bathing medium. The cells were washed in drug-free medium, and 20 min were allowed to elapse before application of the same agonist was repeated, to prevent desensitization of the cells to agonists. Baseline values of PD, Iscc, and R tended to decline with time and successive washes. After administration of agonists, these values were compared with the baseline values, which were taken immediately prior to administration of the agonists. The effects of agonists were expressed as change in PD, Iscc, or R from baseline values. Where blockers of histamine receptors or ion channels were used, the cells were incubated in the presence of the blocker on both apical and basal bathing medium for 20 min prior to addition of histamine. The effect of the blockers of ion channels on PD and Iscc have been shown previously [6].

Measurement of Intracellular Calcium

For measurement of intracellular calcium, approximately 100 µl of cell suspension was placed on 11-mm glass coverslips precoated with laminin (100 µg/ml; Sigma). These were incubated for 24 h to allow the cells to adhere to the glass before being flooded with prewarmed culture medium. The cells were then incubated at 37°C and gassed with 5% CO2/95% air for 5–7 days before being used for intracellular calcium measurement. The cells were incubated in media containing 10 µM Fura 2-AM for 25–60 min for dye loading to occur. Ratio of emission was not influenced by duration of dye loading between these times. The coverslips were then mounted in a small perfusion chamber on the stage of a Nikon Diaphot Epifluor fluorescent microscope fitted with an excitation filter changer and photon counting system (Newcastle Photometric Systems Ltd., Newcastle-upon-Tyne, UK). Single cells or small clumps of cells were visualized and excited alternately every 1.1 sec with light at 350 nm and 380 nm. Light emitted from the cells was measured at wavelengths > 520 nm. The ratio of the emission at the two excitation wavelengths was calculated to give a value related to the intracellular free calcium ([Ca2+]i), which was recorded on a computer screen. Solutions were prewarmed to 37°C and perfused through the chamber at a rate of 3 ml/min. The normal perfusion solution contained 94.7 mM NaCl, 4.78 mM KCl, 1.19 mM KH2PO4, 1.10 mM MgSO4, 5.56 mM glucose, 1.71 mM CaCl2, 4.0 mM NaHCO3. Where antagonists of histamine receptors were used, the cells were perfused with normal solution containing the antagonist for 10 min prior to addition of histamine. Control cells were perfused with normal solution for 10 min prior to addition of histamine. All drugs were tested on 2 coverslips of cells from the oviducts of a given patient and from cells from at least 5 different patients. Calibration of the system has been reported in previous publications from this laboratory [5]. A ratio of 1.0 was found to represent a [Ca2+]i concentration of 109 nM. This is not an absolute method of calibration in that the dye may behave in a different manner inside the cells, and elements such as viscosity and protein binding are unaccounted for; however it does provide an approximation of [Ca2+]i. Results, therefore, are presented in terms of a ratio value.

Measurement of Myosalpingeal Contractions

For measurement of myosalpingeal contractions, 1-cm segments were cut from the oviduct and slit open longitudinally. The segments were suspended by means of threads in an organ bath containing 20 ml Krebs Ringer bicarbonate (as described above) at 37°C, gassed with 5% CO2/95% O2. The segments were attached to an isometric force-displacement transducer (Harvard Instruments, Cambridge, MA) such that contractions of either the circular or longitudinal muscle layers were recorded. Contractions were recorded on a Gallenkamp 2-channel pen recorder, and their frequency and amplitude were analyzed over 10-min periods. Drugs were made up in Krebs Ringer and added in 20-µl volumes in a cumulative manner. Where antagonists of histamine receptors were used, the segments were incubated with Krebs Ringer containing the agonist for 20 min prior to addition of histamine.

Amiloride, tetraethylammonium chloride (TEA), 4-acetamido-4'-isothiocyanostilbene-2.2'-disulfonic acid (SITS), furosemide, verapamil, histamine, PAF, triprolidine, ranitidine, thioperamide maleate, prostaglandin E1, prostaglandin E2, and Fura 2-AM were obtained from Sigma Chemical Co. Leukotriene B4 was obtained from Calbiochem (Nottingham, UK). Dosages of ion channel blockers used in this study were similar to those used in studies on airway, endometrial, kidney, and other cultured epithelia.

Results were analyzed statistically by analysis of variance followed with paired or unpaired Student's t-tests.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Epithelial cells from human oviducts formed a polarized layer that became confluent usually by 5–6 days of culture. The cells formed a rather leaky electrical system; mean values for resting potential difference, short-circuit current, and resistance were 4.5 ± 0.2 mV, -23.5 ± 0.9 µA, and 196.7 ± 6.6 {Omega}/cm2, respectively (n = 99 filters). The filters were used only if values for resistance of at least approximately 100 {Omega}/cm2 could be achieved, this being substantially greater than the 25 {Omega}/cm2 resistance of the filters alone. No effect of stage of menstrual cycle on resting PD and Iscc or on response to agonists was observed.

Effect of Histamine on Electrophysiology of Oviductal Epithelial Cells: Interaction with H1, H2, or H3 Receptors

Histamine (0.1 µM-1 mM) applied to the basal surface of the cells induced a transient, marked increase in PD and Iscc and a small reduction in transepithelial resistance in a dose-dependent manner (Fig. 1). Histamine applied to the apical surface of the cells also induced a transient increase in PD and Iscc but to a lesser degree (Fig. 2). Histamine (10 µM) applied to the apical surface produced increases of 0.52 ± 0.14 mV in PD and 2.96 ± 0.67 µA in Iscc; whereas 10 µM histamine applied to the basal surface of the cells produced increases of 1.6 ± 0.2 mV in PD (p < 0.05 compared with response to apical histamine) and 13.7 ± 1.56 µA in Iscc (p < 0.02 compared with response to apical histamine, n = 12 filters). Pretreatment of the cells with an H1 receptor antagonist (triprolidine, 1 µM, n = 7 filters) antagonized the increases in PD (p < 0.001, Fig. 1) at histamine concentrations between 1–100 µM and Iscc (p < 0.001, 10 µM histamine, p < 0.05, 100 µM histamine) produced by basal application of histamine. Pretreatment with antagonists of H2 (ranitidine, 10 µM, n = 7 filters) or H3 (thioperamide, 1 µM, n = 7 filters) receptors had no effect on increases in PD or Iscc induced by basal histamine. The response of the epithelial cells to histamine applied apically was not significantly reduced by pretreatment with any of the histamine receptor antagonists (Fig. 2).



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  		FIG. 1. Change in transepithelial potential difference with increasing histamine concentration applied to the basal surface of the cells in control (n = 12) cultured human tubal epithelial cells and cells treated with 1 µM triprolidine (n = 6), 10 µM ranitidine (n = 6), or 1 µM thioperamide (n = 6).



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  		FIG. 2. Change in transepithelial potential difference with increasing histamine concentration applied to the apical surface of the cells in control (n = 12) cultured human tubal epithelial cells and cells treated with 1 µM triprolidine (n = 6), 10 µM ranitidine (n = 6), or 1 µM thioperamide (n = 6).

Effect of Ion Channel Blockers on Histamine Action

Pretreatment of the cells with amiloride (100 µM, n = 5 filters), TEA (25 mM, n = 7 filters), or furosemide (100 µM, n = 9 filters) did not affect the response of the cells to histamine applied either apically (data not shown) or basally (Fig. 3). Pretreatment of the cells with 100 µM verapamil (n = 7 filters) reduced responses of the cells to lower doses of histamine applied basally (PD: 1 µM, p < 0.02; 10 µM, p < 0.05, Fig. 4a; Iscc: 10 µM, p < 0.02, data not shown). Pretreatment of the cells with SITS (1 mM, n = 7 filters) reduced the responses to high doses of histamine applied basally (PD: 10 µM, p < 0.05; 100 µM, p < 0.01, Fig. 4b; Iscc: 100 µM, p < 0.01, data not shown). The response to basal or apical histamine was significantly reduced when the cells were incubated in chloride-free medium (n = 5 filters; PD after basal histamine: 1 µM, p < 0.05; 10 µM, p < 0.05; 100 µM, p < 0.01, Fig. 4b. PD after apical histamine: 10 µM, p < 0.01; 100 µM, p < 0.02 data not shown).



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  		FIG. 3. Change in transepithelial potential difference with increasing histamine concentration applied to the basal surface of the cells in control (n = 12) cultured human tubal epithelial cells and cells treated with 100 µM amiloride (n = 6), 100 µM furosemide (n = 8), or 25 mM TEA (n = 7).



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  		FIG. 4. Change in transepithelial potential difference with increasing histamine concentration applied to the basal surface of the cells in a) control (n = 12) cultured human tubal epithelial cells and cells treated with 100 µM verapamil (n = 8) and b) control (n = 12) cultured human tubal epithelial cells, cells treated with 1 mM SITS (n = 9) and cells incubated in chloride-free medium (n = 5).

Effect of PAF on Electrophysiology of Epithelial Cells

PAF (1.9 nM-1.9 µM, n = 8 filters) applied to the apical surface of the cells produced an increase in PD (Fig. 5) and Iscc (p < 0.01 at 190 nM), and a decrease in transepithelial resistance (p < 0.05 at 190 nM). PAF was more effective when applied to the apical surface of the cells than when applied to the basal surface (PD: p < 0.02, Iscc: p < 0.01 at 190 nM PAF).



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  		FIG. 5. Change in transepithelial potential difference in cultured human oviductal epithelial cells with increasing concentration of PAF applied to either the apical surface (n = 8) or the basal surface (n = 7) of the cells.

Effect of Histamine on Intracellular Calcium in Epithelial Cells

Histamine (1–100 µM) produced transient increases in Fura 2-AM fluorescence, indicating an increase in [Ca2+]i (Fig. 6a). Although 10 µM histamine produced a similar response to 1 µM, the response to 100 µM was diminished, probably due to depletion of calcium stores. Pretreatment of the cells with the H1 receptor antagonist, triprolidine (1 µM), greatly reduced or completely inhibited the histamine-induced increase in intracellular calcium (p < 0.001, Figs. 6b and 7). Pretreatment with H2 receptor antagonists—cimetidine (100 µM) or ranitidine (10 µM)—also significantly reduced the histamine-induced increase in [Ca2+]i (p < 0.001). Similarly, pretreatment with H3 receptor antagonist, thioperamide (1 µM), reduced the histamine-stimulated increase in [Ca2+]i (p < 0.001, Fig. 7).



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  		FIG. 6. Recordings of ratio of light emission by Fura 2-AM at excitation wavelengths 350 nm and 380 nm in cultured human oviductal epithelial cells in response to 10 µM histamine in a) control cells and b) cells pretreated with 1 µM triprolidine.



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  		FIG. 7. Change in fluorescence ratio in cultured human tubal epithelial cells perfused with 10 µM histamine only (n = 10) or 10 µM histamine following 10 min perfusion with 1 µM triprolidine (n = 10), 100 µM cimetidine (n = 8), 10 µM ranitidine (n = 9), or 1 µM thioperamide (n = 10).

Effects of PAF, LTB4, PGE1, and PGE2 on Intracellular Calcium

PAF (1–10 nM) failed to increase Fura-2 AM fluorescence in epithelial cells from all but one of 5 patients. Intracellular calcium levels in cultured epithelial cells did not appear to be modulated in the presence of the other inflammatory mediators tested. Thus, no change in fluorescence of Fura 2-AM was detected at any concentrations of the following inflammatory mediators: LTB4 at 10–100 nM; PGE1 and PGE2 at 10 µM.

Effect of Histamine on Oviductal Contractions: Interaction with H1, H2, or H3 Receptors

Histamine (0.1 µM–1 mM) induced a sustained increase in frequency of contraction of both circular (p < 0.05 at 10 µM histamine, n = 10) (Fig. 8) and longitudinal (p < 0.05 at 1 mM histamine, n = 7) muscle layers in the human oviduct. Histamine appeared to be more effective in stimulating the circular muscle layer than the longitudinal layer. In both muscle layers, the increase in frequency was accompanied by a decrease in amplitude of contraction. Pretreatment of the segments with H1 (triprolidine, 1 µM, n = 5) or H3 (thioperamide, 1 µM, n = 5) receptor blockers antagonized the histamine stimulation of contractions in the circular muscle layer (Fig. 8), whereas pretreatment with H2 receptor blocker (ranitidine, 10 µM, n = 6) had no significant effect on the response to histamine. The response of the longitudinal muscle layer to histamine did not appear to be significantly affected by any of the histamine receptor antagonists tested (data not shown).



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  		FIG. 8. Frequency of contraction of circular muscle of human oviduct with increasing histamine concentration in a) control tissue (n = 10) or tissue preincubated with 1 µM triprolidine (n = 5) or 1 µM thioperamide (n = 5) and b) control tissue (n = 10) or tissue preincubated with 10 µM ranitidine (n = 6).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that inflammatory mediators have marked effects on the electrophysiology and intracellular calcium concentration in human oviductal epithelial cells. These effects are comparable with those seen in other epithelia, such as intestinal mucosa. It is possible, therefore, that the actions of inflammatory mediators are similar in inflammatory bowel disease, pelvic inflammatory disease and other inflammatory conditions. In the human, serum histamine concentrations vary from 5 µg/L (0.03 µM) in healthy, nonpregnant women to 25 µg/L (0.14 µM) during severe preeclampsia [25] and 10 µg/L (0.06 µM) during labor. Local tissue concentrations may well be higher.

Previous studies using this culture method for human oviducts have demonstrated that a pure population of epithelial cells is obtained and that the cells form a polarized layer [5, 26]. The oviduct epithelium is considered to be electrically "leaky" [1], with high bi-directional fluxes of ions occurring, probably via paracellular junctions. Resistances above 300 {Omega}/cm2 were rarely achieved.

In secretory epithelia, such as tubal epithelia, cell function is dependent on movement of ions. Chloride ion movements from the basal to apical poles of the cells play a significant role in providing the driving force for fluid movement [27–29]. A low intracellular Na+ concentration is maintained by the Na+/K+-ATPase, with uptake of Cl- via the Na+/K+/2Cl- cotransporter occurring at the basolateral membrane. The apical membrane also becomes permeable to Cl-, which allows the anion to move down an electrochemical gradient. The Cl- ion movements generate an electrical force that drives Na+ paracellularly through the tight junctions between the cells into the lumen, creating a higher luminal osmotic pressure. Water follows the ion movements towards the osmotic equilibrium and thus accumulates in the lumen [29]. Movements of chloride ions from basal to apical have been observed in the epithelium of the oviduct consistent with it being a chloride secretory epithelium [2, 4, 26, 30]. In vascularly perfused rabbit oviduct, inhibition of chloride ion transport decreased vascular to lumen Cl- flux and the rate of fluid secretion [3]. Modulation of transepithelial permeability of most electrolytes and non-electrolytes occurs via alteration in the permeability of the paracellular pathway, and the permeability of the apical and/or the basolateral membranes, resulting in a change in the rate or direction of movement of electrolytes [31]. The basement membrane and the connective tissue layers do not offer a significant barrier to movement of small electrolytes or non-electrolytes.

In the present study, we have shown that histamine influences ion movements in cultured human tubal epithelial cells and therefore may influence tubal secretory function. Transepithelial PD was increased by histamine via activation of H1 receptors. Reduction of the histamine response by pretreatment with ion channel blockers or incubation in chloride-free medium suggested that modulation of the movement of chloride ions and calcium ions may be involved. [Ca2+]i concentrations were increased by doses as low as 1 µM histamine. This effect of histamine appears to be mediated predominately via H1 receptors, but it may also involve activation of H2 and H3 receptors. Histamine was more effective when applied to the basal surface of cultured cells, as would be expected from a blood-born agonist. Histamine was less effective when applied to the apical surface of the cells. It is not known whether, in chronically inflamed oviducts, histamine would be present in the lumen. The lack of antagonism by H1, H2, or H3 receptor antagonists suggests that histamine may act via an alternative mechanism at the apical surface of the cells. Cells grown on glass, as in studies on [Ca2+]i, might not be expected to respond strongly to histamine since only the apical surface would be exposed to the agonist. Single cells, or small clumps of cells, may have small but sufficient areas of the basolateral membrane exposed to the bathing medium to permit response to histamine The rather small number of histamine receptors available under these conditions may explain why all three receptor antagonists were effective in reducing the response to histamine. In epithelial cells from other tissues, histamine has been shown to promote microvascular hyperpermeability by an increase in intracellular calcium [32, 33]. Histamine has been suggested to augment voltage-dependent Ca2+ currents in vascular cells, and activation of Cl- currents may occur secondarily as a result of the elevation of [Ca2+]i [34]. In mammalian small-intestinal epithelia, histamine increases transepithelial potential difference and short circuit current at doses similar to those used in this study [35–37]; and it increases fluid secretion [37]. In rat small intestine, guinea pig ileal mucosa, and porcine distal colon epithelium, the response to histamine is greatly reduced in chloride-free medium or in the presence of the chloride channel blocker, furosemide [36–38], suggesting that Cl- secretion was responsible for the observed changes in electrical properties and fluid secretion. Similar to our findings in oviduct epithelium, the electrical and secretory responses to histamine in rat small intestine were reduced by verapamil, suggesting that the response is Ca2+-dependent. Interestingly, indomethacin also inhibited the response to histamine in rat small intestine, suggesting that histamine induces intestinal secretion by stimulating production of prostaglandins. Prostaglandins E1 and E2 have been shown to modulate chloride secretion in colonic mucosa [39] and in porcine and mouse endometrial epithelium [40, 41]. It is unlikely that histamine acts via prostaglandin production in human oviductal epithelium, since prostaglandins themselves had no effect on the electrophysiology or [Ca2+]i in these cells. Similarly, LTB4 did not influence PD or intracellular calcium. Thus, in pelvic inflammatory disease where oviducts are chronically inflamed, as with inflammatory bowel disease and other inflammatory conditions, histamine may affect tubal function via movement of Ca2+ and Cl-.

Platelet-activating factor produced a marked increase in transepithelial PD when applied to the apical surface of the cells but was considerably less potent when applied to the basal surface. The actions of PAF as an inflammatory mediator may, therefore, be insignificant compared to its role in mediating embryo signaling to the oviduct. Ortiz et al. [42] observed that, in the hamster, embryos were transported to the uterus faster than oocytes. Different rates of transport were observed in the same animals that were inseminated with fertile spermatozoa into one oviduct and infertile spermatozoa into the contralateral oviduct. This suggests that the transport of embryos to the uterus is influenced by local rather than systemic signals [43]. In the hamster, PAF of embryonic origin is the signal that is thought to control transport of the embryos to the uterus [21]. Expression of mRNA encoding the PAF receptor is restricted to the endosalpinx and is most prominent in the subepithelial cells located in the mucosal folds that protrude into the lumen of the oviduct. It is not known whether PAF acts by increasing contractile activity of the myosalpinx, although increased ciliary beat of hamster oviduct ciliated epithelial cells has been observed by addition of physiological concentrations of PAF in vitro [17, 44]. In the rabbit, it is thought to be the beating of the ciliated epithelial cells that propels the embryo towards the uterus [45]. Exogenous PAF, applied to the serosal surface of tissues, has been shown to increase chloride secretion in both small and large intestine of the rat in vitro [46–48] via the stimulation of the Na+/K+/2Cl- cotransporter. PAF also increases fluid secretion via chloride ion secretion in human colonic mucosa [49]. Bumetanide, which inhibits the Na+/K+/2Cl- cotransporter, reduced the effects of PAF. PAF has been shown to increase [Ca2+]i concentrations in cultured hamster or bovine oviduct epithelial cells [17, 22]. Surprisingly, similar concentrations of PAF only increased [Ca2+]i concentrations in human tubal epithelial cells from one patient. The reason for this discrepancy is not clear. It is possible that receptors for PAF, or their coupling to intracellular signal-transduction mechanisms, may not be present in the apical membrane of the epithelial cell throughout the cycle but only immediately postovulation and during early embryo development. If the synthesis or coupling of PAF receptors is controlled by ovarian hormones, epithelial cells from oviducts removed from patients at preovulatory stages of the menstrual cycle may not have received the appropriate hormonal stimulus. In hamsters, mRNA encoding the PAF receptor was observed in oviducts from both pregnant and cycling animals [21]. In the bovine, however, the increase in [Ca2+]i induced by PAF was significantly less in cells from heifers at estrus than in cells from cyclic and pregnant animals [22]. Also, the activity of PAF:acetylhydrolase (PAF:AH) in mouse uterus was found to be regulated by ovarian steroids [50]. Progesterone suppressed PAF:AH activity in the uterus, while estradiol increased its activity. Thus, in an estrogen-dominated tissue, the half-life of PAF would be reduced. Monomeric PAF added to media in the absence of albumin sticks to the surface of glass vessels [51]. It is possible that PAF was ineffective in raising intracellular calcium in our system due to such loss of PAF.

Histamine increased the frequency of contraction of the longitudinal and circular muscle layers of the human oviduct. The amplitude of contraction, however, was often reduced so that, rather than inducing strong contractile activity, histamine action resulted in uncoordinated contractions. Although the role of smooth muscle contractions of the oviduct in gamete and embryo transport is poorly understood, uncoordinated contractions may adversely affect this function. In pelvic inflammatory disease and hydrosalpinx, reduced fertility may be due to high tissue concentrations of histamine and other inflammatory mediators that may result in abnormal fluid production and disruption of normal smooth muscle contractions.

In summary, inflammation of the human oviduct results in reduced fertility. Histamine may, by influencing tubal fluid production, play a role in this reduction of fertility. Histamine, released from mast cells at the site of inflammation, may act on the basal membrane of the epithelial cells and increase calcium influx, thereby increasing [Ca2+]i (Fig. 9). A result of increased [Ca2+]i may be activation of the Cl-/HCO3- antiporter at the basal membrane. Chloride ions transported into the epithelial cell (whilst bicarbonate ions are transported out of the cell) provide the driving force for movement of water into the cell. The extrusion of Cl- ions from the apical surface of the epithelial cell (driving water down the electrochemical gradient into the lumen) may also be influenced by the action of histamine, thus affecting the rate of tubal fluid production. It is also possible that histamine influences the composition of the fluid secreted by the epithelial cell. Histamine and PGE2 may also affect normal myosalpingeal contractions in inflamed oviducts, which may disrupt transport of ova down the oviduct or embryos to the uterus. PAF, in contrast, appears to act predominately at the apical surface of the epithelial cell as a signal from the early embryo. PAF action, perhaps via increased [Ca2+]i and/or Cl- movement in the epithelial cell, ensures normal transport of the developing embryo to the uterus.



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  		FIG. 9. Schematic diagram of the hypothetical action of inflammatory mediators in tubal epithelia and smooth muscle.


   ACKNOWLEDGMENTS
 
We thank Christine Hall and Judith Hawkhead for technical assistance with cell cultures.


   FOOTNOTES
 
1 Supported by the Wellcome Trust, Grant No. 049008/Z/96/Z. Back

2 Correspondence: S.J. Downing, Department of Biology, University of York, PO Box No. 373, York YO21 5YW, UK. FAX: 44 1904 432860; sjd13{at}york.ac.uk Back

Accepted: April 13, 1999.

Received: September 17, 1998.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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