Role of some trace metal ions in placental membrane lipid peroxidation.

The antioxidative and/or pro-oxidative potential of three trace metal ions, namely aluminum (Al), manganese (Mn), and selenium (Se), has been studied. The effect of Al and Mn was found to be anion independent. The pro-oxidative potential of Al was more prominent than its antioxidative potential. This may be due to its redox inert nature. The increase in lipid peroxidation rates in placental syncytiotroblast membranes may contribute to the etiology of aluminum toxicity. Selenium had an antioxidative potential only in the whole-cell homogenate. This appears to be mediated by glutathione peroxidase of which Se is a cofactor. Manganese proved to be the trace metal ion of choice. It decreased the production of thiobarbituric acid reactive substances (TBARS). This may be due to its capacity to quench the superoxide anion and hydroxyl radicals and also due to its chain-breaking capacity. During the present course, ferrous-ascorbate mediated lipid peroxidation has been studied using various combinations of FeSO4 and ascorbic acid. Extrapolating the combined ratio of the individual combination as substrate concentration ([S]) and treating the observed amount of malondialdehyde (MDA) produced equivalent to initial velocity (vi), as in the case of enzymatic studies, the data were treated according to Michaelis-Menten kinetics and the values of kc and Cmax have been calculated.

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.

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.

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.

Effect of sulfasalazine on adaptive and functional changes in intestine of normal and protein-calorie-malnourished rats.

The effects of sulfasalazine (500 mg/kg body weight daily for 35 days) and its subsequent recovery for another 35 and 65 days have been investigated on the intestinal uptake of certain end-product nutrients, viz. glucose, leucine, alanine, and calcium, in normal and protein-calorie malnourished (PCM) male albino rats. Sulfasalazine administration caused a reduction in body weight in PCM animals, while intestinal weight and length as well as protein and nucleic-acid contents were reduced in both normal and PCM animals. Serum proteins also showed a decrease in PCM rats. PCM rats showed elevated levels of glucose, amino acids, and calcium uptake by the intestinal segments, but sulfasalazine feeding inhibited the uptake of nutrients both in normal-fed and malnourished animals. All these changes were found to be reversible after the withdrawal of drug treatment. Sulfasalazine caused a decrease in the Na(+)-dependent (active) glucose uptake as well as the Na(+)-independent (passive) process. The kinetic parameters of glucose uptake indicate that the drug might interfere with the transport/carrier protein of these nutrients, because reduction was observed in maximum uptake velocity (Jmax) of these systems without any change in the affinity constant (Kt).

Effects of embelin, a male antifertility agent, on absorptive and digestive functions of rat intestine.

Oral administration of embelin (75 mg/kg per day, daily for 15 and 30 days) to male rats caused significant elevation in the uptake of D-glucose, L-alanine, L-leucine and calcium in small intestinal segments. Embelin also produced significant increases in intestinal brush border membrane-associated enzymes (sucrase, lactase, maltase, alkaline phosphatase and leucine aminopeptidase) in both intestinal homogenates and partially purified brush border membrane preparations. Significant increases were also noted for microsomal glucose-6-phosphatase and cytosolic lactate dehydrogenase. Increase in brush border membrane-associated total lipids, phospholipids, cholesterol, triacylglycerol, unesterified fatty acids and ganglioside sialic acid were seen but not in the cholesterol/phospholipid molar ratio. All these changes returned to control or near control levels following withdrawal of the drug.

Alterations and reversibility of the effects of gossypol acetic acid on the intestinal uptake of end product nutrients in normal and protein calorie-malnourished male rats.

The effects of gossypol acetic acid, an experimental male antifertility agent, 20 mg/kg body weight daily for 15 days and its subsequent withdrawal have been investigated on the intestinal uptake of certain end product nutrients, namely glucose, leucine, alanine and calcium, in normal and protein-calorie-malnourished (PCM) male albino rats. Gossypol feeding caused a reduction in body weight, intestinal weight and length, protein and nucleic acid contents in both normal and PCM animals. Serum parameters, e.g. total protein, albumin and globulin, also showed a decrease in PCM rats. PCM per se considerably elevated the uptake of nutrients but gossypol feeding inhibited the uptake of nutrients both in normal-fed and malnourished animals. Gossypol caused a decrease in the Na(+)-dependent (active) glucose uptake while the Na(+)-independent (passive) process was not altered. The kinetic parameters of glucose uptake indicate that gossypol might be interfering with the transport/carrier protein of these nutrients as reduction in maximum uptake velocity (Jmax) of these systems was observed without, however, any change in the affinity constant (Kt).

Gossypol-induced inhibition of glucose uptake in human ejaculated spermatozoa may be mediated by lipid peroxidation.

Gossypol, a plant-derived polyphenolic compound known to exert contraceptive actions in men, inhibits 14C-glucose uptake in vitro in human ejaculated spermatozoa. Spermatozoal glucose uptake was found to increase monotonically up to 40 min, and then decreased by 60 min, possibly because of the saturation of the transport loci in the membrane. Gossypol at the concentrations of 5 and 10 microns caused a reduction of both the linear portion of the uptake and the fall afterwards. Gossypol similarly affects both the Na-dependent and -independent glucose uptake. The kinetic parameters of glucose uptake indicate that gossypol might be interfering with the transport/carrier protein as reduction in maximum uptake velocity (Vmax) was observed without any change in the affinity constant (Km). Similarly, gossypol also produced an increase in spermatozoal lipid peroxidation as evidenced by a steep rise in thiobarbituric acid reaction products in the human sperm cells. A significant decrease in total phospholipid level and the individual classes was noted after gossypol addition. Gossypol-induced inhibition of glucose uptake may be related to the generation of lipid peroxides and consequent membrane damage.

Changes in glucose/amino acid/calcium uptake and brush-border membrane-associated enzymes in rat small intestine after the administration of embelin (plant benzoquinone), an antifertility agent.

Administration of Embelin, an experimental antifertility agent, to male rats (20 mg/kg body wt/day, daily for 15 and 30 days), caused an elevation in the uptake of D-glucose, L-alanine, L-leucine, and calcium in the small intestinal segments. An increase was also noted in the intestinal brush border membrane (BBM)-associated enzymes, sucrase, lactase, maltase, alkaline phosphatase, and leucine aminopeptidase in both the intestinal homogenates and partially purified BBM preparations, particularly after 30-day administration of the drug. Embelin treatment also caused a significant increase in the microsomal glucose-6-phosphatase and the cytosolic enzyme, lactate dehydrogenase. In the Embelin-treated animals BBM-associated total lipids, phospholipids, cholesterol, triacylglycerol, unesterified fatty acids, ganglioside-sialic acids as well as the cholesterol/phospholipids molar ratio showed a considerable increase. All these changes in the Embelin-treated animals were restored back to the normal or near normal biochemical makeup when the drug therapy was withdrawn and the animals were allowed to recover for another 15 and 30 days, respectively.

Effect of gossypol on the fertility of male rats.

Thirty male rats were grouped into 5 groups of 6 animals each. Animals in groups II-V were given gossypol at a dose of 5 mg/kg, 10 mg/kg, 20 mg/kg and 40 mg/kg body weight per day for 45 days respectively. Animals of group I served as control. A significant decrease in body weight after administration of 40 mg/kg body weight of gossypol was observed; low doses of gossypol, however did not affect the body weight. Testis, epididymis, prostate and seminal vesicles weights decreased gradually with the increasing doses of gossypol. With the increasing doses of gossypol, a marked decrease in the vas deferens sperm motility was observed. At 40 mg/kg dose there was a total inhibition of sperm motility. Histological studies after 5 mg/kg revealed no apparent sign of degeneration, while after 10 mg/kg dose the changes in the individual cell types were accompanied by overall disorganisation of the germinal epithelium involving displacement of the spermatocytes. The rats treated with 20-40 mg/kg gossypol showed a pronounced deleterious effect on the histological structure of the testis. The drug effect was dose dependent developing sequentially; from the uppermost layer of elongated spermatids affecting round spermatids and finally spermatocytes. Quantitatively the ratios of pachytene spermatocytes: resting spermatocytes, stage 7 spermatids: pachytene spermatocytes, and stage 19 spermatids: stage 7 spermatids and tubular diameter and germinal height decreased significantly. The activities of glucose-6-phosphatase, fructose 1, 6-diphosphatase, glucose-6-phosphate isomerase in testis decreased significantly at high dose (40 mg/kg), while the activity of amylase and glycogen content increased significantly with the increasing doses of gossypol.(ABSTRACT TRUNCATED AT 250 WORDS)

Gossypol inhibition of Ca++ uptake and Ca++-ATPase in human ejaculated spermatozoal plasma membrane vesicles.

Gossypol, a plant-derived polyphenolic compound known to exert contraceptive actions in men, inhibits Ca++-transport and Ca++-activated ATPase in isolated plasma membranes of ejaculated human sperm cells. It also inhibits the membrane bound Mg++- and Na+ + K+-dependent ATPases, 5'-nucleotidase and alkaline phosphatase systems. Ca++-ATPase inhibition by gossypol is non-competitive. It abolishes the discontinuity in Arrhenius expression of temperature dependence of Ca++-ATPase and increases the energy of activation. Phosphatidyl choline and Na+-deoxycholate inhibit Ca++-transport activity in the membrane vesicles. The apparent similarity of Ca++-transport inhibition by gossypol and phosphatidyl choline may indicate the possible capability of this compound to induce changes in the lipid microenvironment of the membranes, wherein the integral proteins operate. Inhibitory effect of gossypol on the plasma membrane Ca++-pump suggests that gossypol may affect sperm motility by a mechanism which is related to the structure and functions of the plasma membrane.

Antispermatogenic effect of embelin, a plant benzoquinone, on male albino rats in vivo and in vitro.

Embelin, the active principle of the seeds of Embelia ribes Burm, has been isolated and the purity established. Daily subcutaneous administration of the compound at a dose of 20 mg/kg body weight to male albino rats for 15 or 30 days revealed an inhibition of: a) epididymal motile sperm count, b) fertility parameters such as pregnancy attainment and litter size, and c) the activities of the enzymes of glycolysis and energy metabolism. These changes were reversible, as seen after 15 and 30 days of recovery. Addition of embelin to epididymal sperm suspensions caused a dose- and duration-dependent inhibition of spermatozoal motility and the activities of the enzymes of carbohydrate metabolism. Light and scanning electron microscopy showed that both in vivo and in vitro treatment with the drug causes profound morphological changes in spermatozoa such as: a) decapitation of the spermatozoal head, b) discontinuity of the outer membranous sheath in the mid-piece and the tail region, and c) alteration in the shape of the cytoplasmic droplet in the tail.

Reversibility of the effects of gossypol acetic acid, an antispermatogenic/antifertility agent on the intestinal structure and functions of male albino rats.

The effects of Gossypol acetic acid (10 mg/kg b. wt. daily for 15 days), an experimental male antifertility agent and its subsequent withdrawal for another 15 days, on the structure and functions of the rat small intestinal tract have been investigated. Gossypol feeding causes a reduction in body weight and intestinal weight, length, protein, and nucleic acid contents. A 27%-50% reduction in the uptake of glucose, alanine, leucine, and calcium is observed after Gossypol feeding which is found to be reversible after 15 days of withdrawal of the drug. Gossypol also causes a significant reduction in the activities of sucrase, lactase, maltase and alkaline phosphatase in the intestinal homogenates as well as in the purified brush border membrane of the microvillus. A decrease in the maximum of apparent enzyme velocity and no change in the substrate affinity constant in these digestive hydrolases are observed on Gossypol treatment. It also causes a shift in the transition temperature in these enzymes and predictably changes the energy of activation both below and above the temperature of transition, although the Arrhenius expression of the temperature dependence still shows proximity, non-linearity, and is parallel to the control group. These changes are reversed on withdrawal of the drug and during the subsequent recovery period. Recovery experiments also show near identical values in kinetic parameters (Kt and Jmax) of 14C-glucose uptake in jejunal segments both in the presence and absence of Na+ ions. Also, no difference is observed between the control and recovery groups with respect to body and intestinal weight, intestinal length, and DNA, RNA, protein, lactate dehydrogenase and glucose-6-phosphate phosphohydrolase values in the intestinal homogenates. Phospholipid, cholesterol and sialic acid levels in both the groups also show nearly identical values. Molecular mechanism of the effects of Gossypol on brush border membrane-bound enzyme/carrier molecules operation is discussed in view of the kinetic and thermodynamic data obtained.

Effects of gossypol acetic acid on the absorptive and digestive functions of rat intestine.

Oral administration of Gossypol acetic acid (10 mg/kg body wt./day, daily for 15 days), an experimental antifertility agent to male rats, caused significant reduction in the uptake of glucose, alanine, leucine and calcium in the small intestinal segments. Gossypol also caused significant decrease in the intestinal brush border membrane--associated enzymes, sucrase, lactase, maltase and alkaline phosphatase. Kinetic analysis indicated that Gossypol decreased the apparent velocity of the disaccharidases while the Km was not altered. It also caused a shift in the transition temperature in these enzymes and predictably changed the energy of activation both below and above the transition temperature, although the Arrhenius expressions of the temperature dependence still showed proximity and were parallel to the control group.

Effect of selected metal ions on the motility and carbohydrate metabolism of ejaculated human spermatozoa.

Zinc, lead and cadmium in the form of chloride salts when added to a standard assay system containing 80 X 10(-6) ejaculated washed human spermatozoa caused a dose and duration-dependent inhibition of their motility. The activity of certain key enzymes of carbohydrate and energy metabolism, viz, glycogen phosphorylase, glucose-6-phosphatase, fructose-1, 6-diphosphatase, glucose-6-phosphate isomerase, amylase, Mg2+- dependent ATPase and lactic and succinic acid dehydrogenases were also found to be inhibited. The order of inhibitory effects of the heavy metals were zinc less than lead less than cadmium. The metal chelating agent, ethylene diamine tetra-acetic acid (EDTA, disodium salt) also interfered with the spermatozoal motility and inhibited the enzyme activities.