W. O. Fu

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.

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.

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)