Electrogenic ion transport in the mouse endometrium: functional aspects of the cultured epithelium.

A primary culture of mouse endometrial epithelium grown on permeable supports was established and the electrogenic ion transport across the endometrial epithelium was studied using the short-circuit current (I(SC)) technique. Enzymatically isolated mouse endometrial cells were immunostained with epithelial cells markers, cytokeratins, indicating an epithelial origin of the culture. Mouse endometrial epithelial cells grown on Millipore filters formed polarized monolayers with junctional complexes as revealed by light and electron microscopy. The cultured monolayers exhibited an average basal I(SC) of 4.6 +/- 0.3 microA/cm2, transepithelial voltage of 2.7 +/- 0.2 mV and transepithelial resistance of 599 +/- 30 omega cm2. The basal current was reduced by 85% in Na+-free solution and 13% in Cl(-)-free solution. The basal current could also be substantially (57.7%) blocked by an apical Na+ channel blocker, amiloride (10 microM), suggesting that Na+ absorption largely contributed to the basal current. Apical addition of Cl- channel blocker, DPC (2 mM), also exhibited an inhibitory effect, 19.4%, on the basal I(SC), indicating minor involvement of Cl- secretion as compared to that of Na+ absorption. The cultured endometrial epithelium also responded to a number of secretagogues including adrenaline and forskolin with increases in the I(SC), which could involve substantial Cl- secretion. The present study has established a culture of mouse endometrial epithelium exhibiting predominantly Na+ absorption under unstimulated condition, and Cl- secretion in response to various secretagogues. This culture may be useful for studying various regulatory mechanisms of electrogenic ion transport across the endometrial epithelium.

Cation and anion channels in rat and human spermatozoa.

Ionic fluxes are thought to be important in the initiating process of gamete interaction such as acrosome reaction. Different populations of ion channels in rat and human spermatozoa were investigated using the planar lipid bilayer technique. Membrane proteins were isolated from rat and human sperm and inserted into lipid bilayer via fusion. We observed K(+) selective and Na(+)-selective channels, as well as divalent permeable cation channels in membrane preparations from rat sperm K+ channels, which were sensitive to the K+ channel blocker, tetraethylammonium (0.1 mM), exhibited a mean single channel conductance of 24 pS. Whereas, larger conductance, 109 pS, was found to be associated with Na+ channels. Low conductance anion channel, 15 pS, was also observed when permeant cations in the bathing solutions were substituted with N-methyl-D-glucamine leaving Cl- as the major permeant ion species. This channel exhibited a slower channel open and closed kinetics when compared to other cation channels. Both cation and anion channels with characteristics similar to that found in rat sperm were also observed in preparations from human sperm. The variety in the types of ion channels observed in rat and human spermatozoa suggests that ion channels may play different roles in sperm physiology and gamete interaction.

Evidence for independent Cl- and HCO3- secretion and involvement of an apical Na(+)-HCO3- cotransporter in cultured rat epididymal epithelia.

Electrogenic chloride and bicarbonate secretion by cultured rat epididymal epithelia was studied using the short-circuit current (ISC) technique. When incubated in normal solution, 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (cpt-cAMP) caused a rise in the ISC, which was attributable to Cl- and HCO3- secretion. Cl- secretion was found to contribute to the initial transient phase, whereas HCO3- secretion contributed to the sustained phase of the response. HCO3- secretion involves a basolaterally placed Na(+)-H+ exchanger and apical anion channel, most probably the cystic fibrosis transmembrane conductance regulator (CFTR). There is also evidence that an apical electrogenic Na(+)-HCO3- cotransporter is involved in HCO3- exit. CFTR accounted for 70% of HCO3- secretion, while the Na(+)-HCO3- cotransporter accounted for 30%. The possibility that the cotransporter may serve as an alternative pathway for HCO3- secretion in cystic fibrosis is discussed.

Different regulatory pathways involved in ATP-stimulated chloride secretion in rat epididymal epithelium.

The regulatory pathways involved in the ATP-stimulated Cl- secretion across rat epididymal epithelium were investigated by the short-circuit current (ISC) technique. Biphasic characteristic was observed in the ISC responded to ATP (0.01-10 microM). Inhibitor of P1 receptor, 8-phenyltheophylline (up to 100 microM), did not have any effect on both phases of the ATP-stimulated ISC. The order of potency for stimulation of the two phases in ISC was ATP > ADP >> AMP, adenosine, consistent with the presence of P2-purinoceptors. Cl- channel blocker, disulfonic acid stilbene (DIDS, 300 microM), only inhibited the first peak of the ATP-stimulated ISC while diphenylamine-dicarboxylic acid (DPC, 1 mM) reduced both, indicating the involvement of different conductance pathways. DIDS was found to have an inhibitory effect on Ca(2+)-activated ISC (induced by ionomycin, 10 microM) but not cAMP-activated ISC (induced by forskolin, 1 microM) which could only be blocked by DPC. Both peaks of the ATP-activated ISC could be significantly inhibited by pretreatment with a Ca(2+)-chelating agent, BAPTA-AM (50 microM). An increase in cellular cAMP content upon stimulation of ATP was measured by radioimmunoassay. No significant increase in cAMP production was observed in cells stimulated with adenosine. The ATP-induced cAMP increase was prevented by pretreatment with BAPTA-AM (100 microM) indicating that cAMP production upon ATP stimulation was secondary to an increase in intracellular Ca2+ concentration. These results indicate that the ATP-stimulated Cl- secretion could be mediated by Ca2+ and cAMP-dependent regulatory pathways giving rise to the biphasic nature of the ATP-induced ISC.

An ATP-activated cation conductance in human epididymal cells.

An outwardly rectifying conductance was observed in primary cultured human epididymal cells under the patch-clamp whole-cell configuration in KCl pipette and bath solutions. Removal of Cl- from intracellular and extracellular solutions did not affect this conductance, but substitution of K+ with N-methyl-D-glucamine in both solutions completely blocked the current. The current magnitude was also found to be affected by intracellular but not extracellular K+ concentrations. The conductance could be inhibited by the cation channel blockers, barium and tetraethylammonium chloride. Single-channel recordings from the same cell population also revealed the presence of a conductance of about 150 pS with voltage dependence and blocker sensitivity similar to those observed for the whole-cell current. This cation conductance was not affected by changes in solution osmolarity but could be activated by extracellular ATP. ATP activation of the conductance could be mimicked by ionomycin and inhibited by BAPTA-AM, an agent that suppresses intracellular free Ca2+. This ATP-regulated cation conductance may be responsible for secreting K+ across the epididymal epithelium, resulting in an observed K+ concentration much higher in the lumen of the epididymis than in the blood.

Regional differences in bioelectrical properties and anion secretion in cultured epithelia from rat and human male excurrent ducts.

Bioelectrical properties and anion secretion in cultured epithelia from different regions of rat and human male excurrent ducts were studied by measuring the short-circuit currents (ISC). In all regions of the rat excurrent duct, Cl- secretion accounts for over 90% of the basal ISC, although the magnitude varied in different regions. Cl- secretion was found to be mediated by a Cl-/HCO3- exchanger, an Na+/H+ exchanger, and an Na+/K+/2Cl- symport located on the basolateral side of the epithelial cells. Forskolin, an activator of adenylate cyclase, and ionomycin, a Ca2+ ionophore, were used to investigate the relative importance of cAMP and Ca2+ as intracellular messengers regulating Cl- secretion in different regions. It was found that in both species, the forskolin-evoked ISC response was larger in the proximal end (efferent duct/caput epididymidis [rat/human, respectively]) than in the distal end (cauda/corpus epididymidis). The response to ionomycin in the rat cauda epididymidis (distal end) was larger than that in the efferent duct (proximal end); on the other hand, no significant difference in the ionomycin-induced ISC was observed in the caput and the corpus regions from the human epididymis. Our results indicate that while the cAMP- and Ca(2+)-dependent pathways are both involved in regulating Cl- secretion in all regions along the male excurrent ducts in both species, a regional difference exists with respect to the relative importance of the two regulatory pathways involved in Cl- secretion along the male reproductive tract.

Adrenergic receptors on cultured rat epididymal cells: regulation of Cl- conductances.

Adrenergic regulation of epididymal Cl- currents was studied by the whole-cell patch-clamp technique using various alpha- and beta-receptor agonists and antagonists in primary cultured rat cauda epididymal cells. Cl- currents could be activated with varying frequency by noradrenaline (primarily alpha- and beta 1-adrenoceptor-selective agonist, 1-5 microM), isoprenaline (nonselective beta-adrenoceptor agonist, 5 microM), salbutamol (beta 2-adrenoceptor-selective agonist, 2 microM), and phenylephrine (alpha 1-adrenoceptor-selective agonist, 1-2 microM). Noradrenaline alone elicited Cl- current activation in 85% of the cells examined. In the presence of phentolamine (nonselective alpha-adrenoceptor antagonist, 15 microM), noradrenaline elicited Cl- current activation in 63% of the cells examined, whereas noradrenaline-induced activation was observed in 33% of the cells examined in the presence of both atenolol and butoxamine (beta 1- and beta 2-adrenoceptor antagonists, respectively, 10 microM). In 27% of single cells examined, a second current activation in response to salbutamol was observed after the first response to phenylephrine. When the order of stimuli was reversed, dual activation was also observed in 22% of the single cells examined, indicating the presence of both alpha- and beta-adrenoceptors in single epididymal cells. Profiles of time- and voltage-dependent Cl- current upon activation by different adrenoceptor agonists exhibited characteristics similar to those previously reported for Ca2+ and cAMP-activated Cl- currents, suggesting that regulation of epididymal Cl- conductances could be mediated by different adrenoceptor subtypes involving Ca2+ and cAMP as intracellular second messengers.

The effect of [Arg8]vasopressin on electrogenic chloride secretion in cultured rat epididymal epithelia.

Primary cultured rat efferent ductal epithelia and cauda epididymal epithelial were mounted in Ussing chambers to study the effect of arginine vasopressin (AVP) on chloride secretion in the male excurrent duct. The regional differences in the signal transduction pathways involved were also investigated. In both the efferent duct and the cauda epididymidis, basolateral addition of AVP resulted in a dose-dependent increase in the short-circuit current (Isc), which was mediated via V1 receptor. Replacement of ambient Cl- with gluconate or pretreatment of a Cl- channel blocker, diphenylamine-2-carboxylate (apical, 1 mM), completely abolished the response, whereas addition of amiloride had no effect on the Isc. Pretreating the epithelia of the efferent duct with indomethacin (apical, 5 microM) or forskolin (basolateral, 1 microM), but not thapsigargin (apical, 1 microM) or trifluoperazine (apical, 20 microM), significantly inhibited the AVP response (P < 0.001). By comparison, pretreating the epithelia of the cauda epididymidis with any of the four agents significantly reduced the AVP-evoked response. These results suggested that the stimulation of chloride secretion by AVP in the efferent duct and the cauda epididymidis is mediated by prostaglandin synthesis and involves adenosine 3',5'-cyclic monophosphate (cAMP) as a second messenger. In the cauda epididymidis, calcium, in addition to cAMP, may play a role in mediating the AVP-induced response.

Swelling-induced anion and cation conductances in human epididymal cells.

1. Activation of both anion and cation conductances was observed in primary cultured human epididymal cells during osmotic swelling under the patch-clamp whole-cell configuration. The swelling-induced anion conductance was 25.66 +/- 4.70 nS and the cation conductance was 7.35 +/- 1.40 nS. The permeability ratio of K+ to Cl- (PK/PCl) was calculated to be 0.40. Known anion or cation channel blockers could inhibit both conductances simultaneously. 2. When the major permeant ion species in the pipette and bath solution was Cl-, the mean conductance was found to be 17.06 +/- 1.8 nS, significantly smaller than that obtained in the presence of intracellular K+, 25.66 +/- 4.70 nS (P < 0.05). No significant current activation was observed when solutions containing only K+ as the permeant ion were used. 3. When the anionic amino acids glutamate and aspartate were used to replace extracellular Cl-, the permeability ratios were calculated to be PGlut/PCl = 0.20 and PAsp/PCl = 0.17. 4. The cation conductance was found to be non-selective since its permeability to other cations such as Na+ and choline, an organic compound highly concentrated in epididymal fluid, was similar to that of K+. 5. Regulatory volume decrease (RVD) was observed after initial osmotic swelling; this could be inhibited by either anion or cation channel blockers. 6. The results of this study suggest that both anion and cation conductances are activated during cellular swelling, and indicate the existence of an interdependent relationship between the swelling-induced cation and anion conductances. Both swelling-induced cation and anion conductances are involved in the volume regulatory process and may be responsible for transporting amino acids or organic compounds in human epididymal cells.

Characterization of a swelling-induced chloride conductance in cultured rat epididymal cells.

Swelling-induced Cl- conductance in cultured rat epididymal cells was characterized using whole cell patch-clamp techniques. Activation of whole cell current with an outwardly rectifying current-potential relationship was observed in cells exposed to hyposmotic solutions. This current was determined, from the observed current-reversal potentials at different Cl- concentrations, to be Cl- selective. The anion selectivity sequence of the swelling-induced Cl- conductance was I- approximately NO3- approximately Br- > Cl- > 2-(N-morpholino)ethanesulfonic acid. The swelling-induced Cl- conductance was reversibly inhibited by different Cl- channel blockers. Unlike diphenylamine-2-carboxylate or 5-nitro-2-(3-phenylpropylamino)-benzoate, which showed voltage-independent blockade, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid showed a marked voltage-dependent blockade of the volume-sensitive Cl- current, with a greater effect at depolarizing voltages. The swelling-induced Cl- conductance appeared to be different from the Ca(2+)- or adenosine 3',5'-cyclic monophosphate-activated Cl- conductances on the basis of the following observations: 1) swelling-induced current activation was seen even in the presence of kinase inhibitor (H-8) or absence of external free Ca2+, and 2) further increase in current activation could be produced by swelling after Ca(2+)- or adenosine 3',5'-cyclic monophosphate-induced current activation. The swelling-induced Cl- conductance may be involved in regulating epithelial cell volume as well as serving other important epididymal functions such as facilitating transepithelial secretion of organic compounds.

Properties of cAMP-dependent and Ca(2+)-dependent whole cell Cl- conductances in rat epididymal cells.

Single rat epididymal cell studied under whole cell patch-clamp condition responded to 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) (500 microM) and to ionomycin (1 microM) by an increase in whole cell conductance. A major part of the stimulated current was carried by Cl-, although a small part was due to nonselective cation current. After elimination of the cation current component by using impermeant cation, the cells revealed different Cl- conductance properties in response to adenosine 3',5'-cyclic monophosphate (cAMP) and ionomycin. The cAMP-stimulated Cl- conductance was independent of time and voltage and showed a linear current-voltage relationship. The anion permselectivity was NO3- > Br- > Cl- approximately I- >> SO(4)2-. The ionomycin-stimulated Cl- conductance showed marked time and voltage dependency. In contrast to the cAMP-induced anion permselectivity, the ionomycin-induced anion permselectivity was I- > Br- approximately NO3- > Cl- >> SO(4)2-. These results indicate that the epididymal epithelial cells exhibit different Cl- conductances sensitive to cAMP and Ca2+. The cAMP-activated conductance has properties resembling the type associated with the cystic fibrosis transmembrane conductance regulator found in cystic fibrosis-affected epithelia. This finding supports the notion that the epididymis is a cystic fibrosis epithelium.

Electrophysiological studies of anion secretion in cultured human epididymal cells.

1. Primary monolayer cultures from adult human epididymis were grown on Petri dishes and previous supports. The epithelia so formed were used for whole-cell patch clamp recording and short-circuit current (ISC) measurement. 2. After 50 days of culture, the cells formed a tight epithelium with transepithelial potential of 5.5 +/- 1.3 mV (mean +/- S.E.M.., n = 16), apical side negative, and a basal ISC of 6.9 +/- 0.9 microA cm-2 (mean +/- S.E.M., n = 16). 3. Adrenaline, when added to the basolateral side, at a concentration of 0.23 mumol l-1 increased the ISC by 3.0 +/- 1.2 microA cm-2 (mean +/- S.E.M., n = 4). This increase was blockable by diphenylamine-2-carboxylate (DPC, 1 mmol l-1). Forskolin (10 mumol l-1) also evoked a similar response to adrenaline. 4. In whole-cell patch clamp experiment, the resting membrane potential of the cells after dialysis with pipette solution containing 135 mmol l-1 KCl was found to be -30 +/- 14 mV (mean +/- S.E.M., n = 15). 5. About 90% of the cells successfully forming patches responded to 1 mumol l-1 adrenaline by an increase in inward current at -70 mV holding potential (delta I = -1600 +/- 900 pA, mean +/- S.E.M., n = 15). This increase in current was accompanied by a shift in reversal potential to -2 +/- 1 mV (mean +/- S.E.M., n = 15). 6. The adrenaline-induced inward current was found to be blockable by the Cl- channel blocker, DPC (0.25 mmol l-1). Ion substitution experiments showed that the adrenaline-evoked current was carried mainly by Cl-. 7. The effect of adrenaline on the whole-cell current was found to be mimicked by forskolin and could be abolished by including GDP beta S or a protein kinase A inhibitor in the pipette solution. Propranolol, but not phentolamine, completely abolished the effect of adrenaline. 8. Inclusion of 20 mmol l-1 EGTA or 2 mmol l-1 BAPTA + 100 mumol l-1 TMB-8 (to inhibit intracellular Ca2+ release) in the pipette did not seem to have any marked effect on adrenaline-evoked whole-cell current. Lowering the pipette Ca2+ concentration to 1 nmol l-1 or raising it to 10 mumol l-1 had no effect on the whole-cell current response to adrenaline. 9. This study shows that adrenaline stimulates Cl- secretion in cultured human epididymal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of angiotensins on electrogenic anion transport in monolayer cultures of rat epididymis.

Confluent monolayers cultured from the rat cauda epididymidis have been shown to respond to angiotensin I (AI) and angiotensin II (AII) when studied under short-circuit conditions and bathed on both sides with Krebs-Henseleit solution. Both the decapeptide AI and the octapeptide AII elicited transient increases in short-circuit current (SCC) when added to the basolateral as well as to the apical surfaces, with the effect of basolateral application greater than that of apical application. The maximal responses produced by AI and AII were similar with median effective concentrations of 20 to 80 nmol/l. The increase in SCC by AII was dependent upon extracellular Cl- and was inhibited by addition of a Cl- channel blocker, diphenylamine 2-carboxylate, to the apical surface. These patterns of activity suggest that the SCC responses to angiotensins result from electrogenic chloride secretion. Pretreating the monolayers with captopril (100 nmol/l), an angiotensin-converting enzyme (ACE) inhibitor, reduced the response to basolateral application of AI, but completely abolished the response to AI added apically. These results suggest that the response to apical addition of AI was due to conversion of AI to AII which interacts with apical angiotensin receptors. This conversion was mediated by ACE which has been detected in epididymal monolayers. Of the endogenous ACE activity, 86% was found to be inhibited by captopril (100 nmol/l). Responses of the epididymal monolayers to angiotensins were mediated by specific angiotensin receptors. [Sar1,Ile8]-AII, a specific antagonist of the AII receptor, completely inhibited the responses to AI and AII but had no effect on the responses to bradykinin and endothelin.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelin stimulates short circuit current in a cultured epithelium.

1. Endothelin, a novel potent vasoconstrictor peptide produced by vascular endothelial cells stimulated anion secretion by a cultured secretory epithelium derived from the rat epididymis as measured by changes in short-circuit current (SCC). 2. Stimulation of the SCC was observed when endothelin was added to the basolateral or the apical side of the epithelium. The response to basolateral application was greater than that to apical application. The EC50 values were found to be 1.3 nM and 3.0 nM for basolateral and apical application, respectively. These values were about half an order to one order of magnitude higher than that required for its vasoconstrictor action. 3. The stimulation of SCC by endothelin was accompanied by a rise in the intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) content in epididymal monolayers. 4. The effect of endothelin on SCC was mediated through an increase in prostaglandin synthesis by the tissues and was not blocked by an antagonist of angiotensin II (Sar1 Ile8 angiotensin II) or of adrenaline (propranolol). 5. It is speculated that endogenous endothelin plays an important role in the control of water and electrolyte transport in the epididymis.

Antifertility effects of some sulphonamides and related compounds and their accumulation in the epididymides of male rats.

Nineteen sulphonamides and related drugs were screened for their antifertility effects in male rats. They were suspended in corn oil and fed orally to rats at 10 times the human dose for a period of 6 months. Of the 19 compounds tested, sulphamethazine, sulphapyridine, dapsone, sulphamethoxypyridazine, sulphaguanidine, sulphathizole, sulphamerazine and sulphadimethoxine reduced fecundity of male rats to 34.3, 37.6, 38.3, 53.6, 55.6, 58, 75 and 78.6% of control, respectively. The fall in fecundity was due to a reduction in the number of embryos compared with the number of corpora lutea per pregnant female, and, in some cases, was associated with a fall in epididymal sperm concentration and motility. Some of these compounds accumulated in the cauda epididymidis at concentrations equal to or higher than the free drug concentrations in the blood. It is proposed that the antifertility effects of some of these compounds may in part be mediated through a direct effect on epididymal stored spermatozoa, hence compromising some processes vital for fertilization.

Gossypol does not affect testicular fluid secretion in rats.

Male rats fed with gossypol acetic acid at a dose rate of 20 mg/kg/day for 16 weeks were rendered infertile. Spermatozoa flushed out from the cauda epididymides completely lost their capacity to initiate forward motility. However, the rate of testicular fluid secretion measured by the efferent duct ligation technique was not affected by gossypol treatment. The sodium and potassium concentrations of the secreted fluid were found to be unchanged. It is concluded that at low antifertility doses, gossypol disrupts spermatogenic elements in the testis without affecting fluid secretion by the Sertoli cells.

Restricted entry of an anti-rat epididymal protein IgG into the rat epididymis.

A rat epididymal protein (MW 32000) was isolated and purified from the rat caudal epididymal fluid. Mono-specific antiserum against this protein was raised in rabbits, purified and labelled with 125I. The labelled IgG was infused intravenously into anaesthetized male rats, and the transfer of the labelled IgG across the luminally perfused cauda epididymidis was studied. It was found that during the 2 h infusion period, radioactivity in blood rose, but no radioactivity could be detected in the perfusates, irrespective of whether the epididymis was perfused with Krebs bicarbonate solution or a solution which resembled the rat caudal fluid in ionic composition. In some experiments, rats were given a single intravenous injection of labelled IgG and radioactivity in the epididymal content was measured 10 days later. It was found that despite a high IgG level in blood and liver, no radioactivity could be detected in epididymal fluid and sperm. It is concluded that the blood-epididymis barrier restricts the passage from blood to lumen, an immunoglobulin directed against an epididymal protein.