Ca(2+)-transport across brush border and basal surfaces of human term [correction of terms] placenta.

Brush border microvillous (BBM) and basal cell membranes (BCM) were isolated from syncytiotrophoblast of human term placenta by homogenization, sonication, prolonged stirring and differential centrifugation. Uptake of 45Ca(2+)-CaCl2 in membrane vesicles in morpholino propane sulphonic acid (MOPS) buffer was studied up to 60 min. Maximum uptake of the radioisotope was recorded at 10 and 15 min of incubation of the BBM and BCM, respectively. Radioisotopic uptake was also dependent on the Ca(2+)-concentration, linear up to 3 mu mole and then assuming hyperbolic substrate saturation kinetics. The Lineweaver-Burk transformation of the data gave Kt value for BBM and BCM, 0.85 and 1.08 microM, respectively while the Vmax of uptake (Jmax) in the same were 105.26 and 188.68 pmole Ca2+/microgram protein/20 min. Ca(2+)-Uptake in placental BBM and BCM vesicles was inhibited by two Ca(2+)-channel blockers, nifedipine and verapamil to as much as 50% while Ca(2+)-ionophore A23187 enhanced the uptake process significantly.

Isolation and characterization of the basal cell membranes of the human term placenta.

A relatively simple and rapid method is described for the isolation of basal cell membranes (BCM) from the human placenta at term which showed considerable improvement in the yield, purity and membrane characteristics as compared to the earlier described methods. The method is based on thorough washings of the syncytium in balanced salt solution, selective grinding, hypotonic lysis, sonication, incubation with EDTA and then more conventional differential centrifugation and ultracentrifugation. The isolated material showed smooth surfaced vesicular structure of various sizes as revealed by both positive and negative staining and transmission electron microscopic analysis. The membrane was highly enriched in Na+/K+, Ca2+ and Mg2+ dependent ATPase activities while the cross contamination with brush border surfaces was low as revealed by the marker enzyme assays specific for the brush border membrane (BBM) such as the disaccharide hydrolases, aminopeptidase and alkaline phosphatase. The membrane showed a relatively low lipid/protein ratio and the lipid composition represented by a variety of phospholipids (phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin, phosphatidyl inositol and phosphatidyl serine), neutral lipids (cholesterol, triacyl glycerol, free fatty acids) and glycosphingolipids (ganglioside, cerebroside and sulfatide). It also contained plasmalogens. On SDS-PAGE analysis and Coomassie blue staining reaction, the isolated membrane showed 14 major bands with as many minor ones with a molecular weight ranging between 30-110 kDa.

Transport of glycine in the brush border and basal cell membrane vesicles of the human term placenta.

Active glycine transport was demonstrated in microvillous (maternal-facing, BBM) and basal (fetal-facing, BCM) plasma membranes of the human term placental syncytiotrophoblast. The kinetic studies showed that the amino acid had a distinct overshoot at 1 min in BBM and 3 min in BCM vesicles while a steady state rate was achieved in approx 5 min in both the vesicles. Glycine transport is highly ion-specific and its dependency on Na+ can not be satisfied by replacing with other monovalent cation. Cl- is also implicated in the generation of the electrochemical gradient and replacement of Cl- with SO4(2-) anions failed to stimulate the transport process. The transport process was saturable with external glycine which exhibited rectangular hyperbolic kinetics typical of a mediated movement. The calculated kt and Jmax from the linear transformation of the data were 6.67 & 4 mM and 294 & 263 nmoles glycine. mg protein-1.min-1 in the BBM and BCM vesicles, respectively. The glycine transport was inhibited by a number of other amino acids which are known to be transported through the A and ASC systems. The glycine transport system may be dependent on multiple pathways such as the A, ASC or Gly which is a variant of pathway A. Glycine transport was inhibited by ouabain, a known Na+/K+ -ATPase inhibitor, in the BCM vesicles but not in the BBM system. Nicotine, insulin, sodium fluoride and sodium arsenate were inhibitors for both the vesicles.

Placental membrane transport: leucine transport across the brush border and basal cell membrane surfaces.

Brush border (microvillous) plasma membranes (BBM) and the basal surfaces (BCM) from the syncytiotrophoblast of human term placenta were prepared by a method of sonication, dialysis and differential centrifugation in specific buffer systems. Such plasma membranes formed closed, osmotically active, right-side-out vesicles in which amino acid transport could be studied unidirectionally by a carefully designed membrane filtration assay under reduced pressure. In such vesicles, L-leucine (1 mM) was found to be transported in a time-dependent manner, peak accumulation being attained at 45 s in both BBM and BCM. The accumulation of L-leucine in the vesicles was dependent on an inward NaC1 gradient, as replacing the Na+ with K+, Li+ and choline, or replacing the C1- with S0(2-4) failed to influence the amino acid movement. Leucine transport in the vesicles also appeared to be dependent on the substrate concentration, indicating saturation at a higher concentration. The transport process showed a k(t) (affinity constant) of 3.85 and 6.67 mM, while the values recorded for the J max (maximum apparent initial velocity) were 270.27 and 384.62 nmol/mg protein-1 per min in the BBM and BCM respectively. Leucine transport was inhibited by a number of amino acids, among which amino isobutyric acid (AIB) produced the maximum inhibition. The k(i) inhibition constant) for different amino acids has also been listed. Lysine showed the least k(i) value, thus showing it to be the most inhibitory compound. These findings are discussed in relation to the mechanism and regulation of transplacental amino acid transfer.

Calcium channel antagonist verapamil modulates human spermatozoal functions.

The effect of calcium channel blocker, verapamil (0.5-50 microM), has been studied in vitro in relation to certain spermatozoal functions in human ejaculates. Disruptive changes in the head and tail region of the spermatozoa, separation of heads from tails and coiling of the tail were observed. Motility was considerably reduced, while the pattern of motility also changed from rapid, linear progression to slow or sluggish linear or non-linear movement and finally to non-progressive motility, or even immotility. Verapamil significantly inhibited the influx of extracellular Ca2+. The study of kinetic effects further revealed a reduction in the maximum uptake velocity, but no change in the apparent substrate affinity constant. A highly significant decrease in Ca(2+)-dependent ATPase activity was also noted. In order to see whether this drug had any cytotoxic effect, presumably through lipid peroxides, thiobarbituric acid-reactive substances were measured. Verapamil produced an increase in the formation and release of malonyldialdehyde. The level of membrane cholesterol and phospholipid in the spermatozoa was also lowered considerably. The potential of such a calcium channel blocking agent in the designing of a male contraceptive programme is discussed.

The effect of nifedipine, a calcium channel blocker, on human spermatozoal functions.

In vitro addition of 0.1-100 microns of a calcium channel blocker, Nifedipine, resulted in a significant non-competitive inhibition in the uptake of Ca++. The activity of Ca(++)-dependent ATPase enzyme was also decreased. Motility of the spermatozoa was significantly arrested following incubation with different doses of the drug at 37 degrees C for different durations. The pattern of motility changed within two hours from rapid and linear progression to slow or sluggish linear or non-linear movement and finally to non-progressive motility or even immotility. Scanning electron microscopic studies revealed disruptive changes in the head as well as tail region and coiling of spermatozoa after Nifedipine treatment. An increase in the formation and release of malonyldialdehyde was observed following Nifedipine addition in a dose-dependent fashion. The membrane cholesterol and phospholipid contents were considerably lowered in the treated samples. The potential of such calcium channel blocking agent in the designing of male contraceptive programme is discussed.

PIP: In vitro addition of 0.1-100 mcM of a calcium channel blocker, nifedipine, resulted in a significant non-competitive inhibition in the uptake of Ca++. The activity of Ca-dependent ATPase was also decreased. Motility of the spermatozoa was significantly arrested following incubation with different doses of the drug at 37 degrees Celsius for different durations. The pattern of motility changed within 2 hours from rapid and linear progression to slow or sluggish linear or non-linear movement and finally to non-progressive motility or even immotility. Scanning electron microscopic studies revealed disruptive changes in the head as well as tail region and coiling of spermatozoa after nifedipine treatment. An increase in the formation and release of malonyldialdehyde was observed following nifedipine addition in a dose-dependent fashion. The membrane cholesterol and phospholipid contents were considerably lowered in the treated samples. The potential of such calcium channel blocking agent in the designing of a male contraceptive program is discussed.