p-aminohippurate uptake by syncytial microvillous membrane vesicles of human term placenta

The mechanism of uptake of p-aminohippurate (PAH) by syncytial microvillous membrane vesicles of human term placenta was investigated. Initial PAH uptake and efflux were increased in the presence of a pH-gradient and a Cl(-)-gradient, respectively. Forced negative and positive membrane potentials did not influence the uptake, which indicated that the transport is not electrogenic. The pH-dependent increase is probably the result of a higher rate of diffusion due to a lower degree of dissociation of PAH. Because several organic anions failed to transstimulate PAH uptake and FCCP did not decrease the uptake in the presence of an inwardly directed H(+)-gradient, ruling out a PAH/OH- antiport, an anion exchange system does not appear to be present in these membranes. Since electrogenicity and anion exchange seem not to be involved in the Cl(-)-dependent increase, an allosteric effect of Cl- on the transporter might be possible. Various organic anions were able to inhibit pH-stimulated PAH uptake significantly. Kinetic analysis of the probenecid sensitive part of uptake provided further evidence for mediated transport of PAH (Km = 7.4 +/- 2.6 mM and Vmax = 2.0 +/- 0.4 nmol/mg/15 s). Non-inhibitable diffusion accounted for the main part of total transport. Concentration dependent inhibition of PAH transport by probenecid showed a Ki of 2.5 +/- 0.9 mM. It is concluded that human placental syncytial microvillous membrane vesicles possess a low affinity transport mechanism for PAH with low specificity. The importance of this system, for placental excretion of anionic drugs, will depend on the intrasyncytial concentration of these drugs, caused by the transport across the basal membrane.     

Constitutive and induced cytokine production by human placenta and amniotic membrane at term

Results of our previous study on the immunity of human placenta and amniotic membranes revealed that in majority of cases these organs present constitutive non-specific antiviral immunity in the organ culture (OC) system. It is possible that interferons (IFNs), tumour necrosis factors (TNFs) and interleukin 6 (IL-6) may be responsible for the antiviral effect. Here, the constitutive and lipopolysaccharide (LPS)-induced production of these cytokines and, additionally, interleukin 10 (IL-10) were determined in OC of chorionic villi, decidua and amniotic membranes. Significant amounts of constitutive TNF-alpha (2-64 U/ml), IL-6 (200-12,000 U/ml) and IL-10 (1-70 ng/ml) were detected in the maternal decidua and chorionic villi of placenta. Amniotic membranes produced lower concentrations of the cytokines. LPS increased the production of cytokines from two- to eightfold. In contrast, activity of IFN released spontaneously was found only in four of 50 placentae and amniotic membranes. LPS and Newcastle disease virus (NDV) induced IFN production in the OC system. However, the increase of IFN after induction was also very small (up to 32 U/ml). Individual differentiation in the cytokines production was observed among placentas and amniotic membranes. TNF was identified as type alpha with addition TNF-beta, IFN as type alpha, beta and gamma.     

Palmitoyl-CoA stimulates cellular uptake and plasma membrane binding of carnitine

Carnitine cellular uptake and plasma membrane binding was investigated in S49 lymphoma cells. Palmitoyl-CoA was found to increase membrane binding of carnitine from 506 +/- 48 to 8,690 +/- 235 pmol/mg membrane protein. Palmitate and CoA acted synergistically and increased carnitine binding to plasma membranes but could not replace palmitoyl-CoA. The effect of palmitoyl-CoA on membrane binding of carnitine was maximal at 10 microM and required the presence of ATP. Palmitoyl-CoA increased the cellular uptake rate of carnitine from 181 +/- 5 to 884 +/- 25 amol/cell and h-1. We conclude that palmitoyl-CoA is a major regulator of cellular uptake of carnitine and, based on quantitative estimations, that the carnitine carrier binds more than one carnitine molecule.     

Calcium and magnesium deficiency-induced atrophy of muscle and calcium accumulation in the spinal cord

Four consecutive patients with mycosis fungoides received cyclic chemotherapy using cyclophosphamide, vincristine, and prednisone (COP). In one case, there was complete remission of disease for 15 months. In another, there was 50% regression of tumors with healing of the ulcerated surfaces. The third patient showed complete clearing of scaling and redness of the skin. In the fourth patient, the lesions progressed.     

Evidence for Mg2, ATP-dependent accumulation of Ca2+ by the intracellular non-mitochondrial calcium store in uterine smooth muscle cells

Using carbachol contracture as the experimental model for testing the properties of the intracellular calcium store in intact tissue and 45Ca2+ accumulation in the chemically skinned by digitonin smooth muscle cells isolated from oestrogen-dominated rat uterus the evidence for the presence of Mg2+, ATP-dependent Ca2+ pump in the non-mitochondrial store has been found which is supposed to play a key role in the process of refilling' of the store on the cytoplasmic level. The experiments performed on intact muscle showed that the functional activity of the carbachol-releasable Ca2+ store is critically dependent on Ca2+ entry. It is found that Ca2+ entry via voltage operated Ca2+ channels or on the Na(+)-Ca2+ exchange was needed to refill the store in this tissue. However, when Ca2+ extrusion systems located in the plasma membrane were inhibited by La3+, the store retained its ability to discharge and reaccumulate Ca2+ released on the regular basis suggesting the presence of the energy-dependent Ca2+ accumulating system in the store. The process of the store refilling was totally inhibited by cyclopiazonic acid. Chemically skinned uterine smooth muscle cells demonstrated the presence of Mg2+, ATP-dependent accumulation of Ca2+ in the non-mitochondrial (ruthenium red insensitive) intracellular store(s) potentiated by Ca(2+)-precipitating anions (potassium oxalate and phosphate), in a time- and concentration dependent way which was inhibited by Ca(2+)-ionophore A 23187 (5 microM) and cyclopiazonic acid with Ki = 0.4 microM. It is suggested that in the uterine smooth muscle of the oestrogen-dominated rats, nonmitochondrial receptor-operated intracellular calcium store is represented by endoplasmic reticulum.     

Calcium and membrane binding properties of bovine neurocalcin delta expressed in Escherichia coli

Neurocalcins are brain-specific proteins that belong to a new subclass of the EF-hand superfamily of calcium binding proteins, defined by the photoreceptor cell-specific protein, recoverin. Recoverin, which regulates the desensitization of photo-excited rhodopsin, is myristoylated and exhibits a calcium-myristoyl switch. Like recoverin, neurocalcins have a signal for N-myristoylation and possess four EF-hands, although the first one lacks some residues critical for calcium binding. In this work, I have examined the calcium and membrane binding properties of recombinant myristoylated and unmyristoylated neurocalcin delta. I show that neurocalcin, like recoverin, binds to biological membranes in a calcium- and myristoyl-dependent manner. Both myristoylated and unmyristoylated proteins bind three calcium ions. However, the unmyristoylated form exhibits a higher affinity for calcium than the myristoylated protein but shows a lower cooperativity in binding calcium. These data support the model for the calcium-myristoyl switch mechanism proposed for recoverin (Zozulya, S., and Stryer, L. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11569-11573; Dizhoor, A. M., Chen, C. K., Olshevskaya, E., Sinelnikova, V. V., and Hurley, J. B. (1993) Science 259, 829-832). Using point mutations, I have investigated the relative importance of each of the three functional EF hands (EF2, EF3, and EF4) in the calcium and membrane binding properties of neurocalcin. Calcium and membrane binding properties of the mutant-myristoylated proteins suggest that binding of calcium to EF2 is critical in triggering the binding of the protein to membranes.     

The plasma membrane of human placenta. Isolation of microvillus membrane and characterization of protein and glycoprotein subunits

A microvillus plasma membrane-enriched fraction of human placenta was obtained by a combination of differential, isopycnic, and rate-zonal centrifugation techniques. Assays for enzyme markers from mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, and plasma membrane indicated a relative enrichment of plasma membrane between 10- and 20-fold over the most prominent contaminating enzyme markers. Electron microscopy verified the microvillus ultrastructure of the isolated placental membrane and the lack of significant contamination by identifiable organelles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the microvillus membrane fraction revealed a protein and glycoprotein subunit composition. There were 16 major protein subunits and 10 major glycoprotein subunits, and apparent molecular weights are assigned to these subunits. 32P-labeling of the microvillus membrane-associated alkaline phosphatase indicated that this enzyme is one of the major glycoproteins of the human placental microvillus membrane.     

The cardiac content of sarcoplasmic reticulum in the rat determined by calcium uptake rate, calcium oxalate capacity, ryanodine binding and thapsigargin titration

The amount of cardiac sarcoplasmic reticulum in rat hearts was estimated by comparing marker activities in the isolated SR fraction with their activities in the homogenate. Four distinguishable markers were measured: the oxalate-supported rate of calcium uptake, the calcium oxalate capacity, 3H-ryanodine binding and the thapsigargin equivalents. The calcium uptake rate and capacity and thapsigargin equivalents were determined in the presence and absence of SR Ca2+ channel blockade with high concentrations of ryanodine. All of these activities are believed to be located only in the SR. However, the calculation of the heart content of SR was somewhat different for the four markers. The calcium uptake rate gave 8.4 mg SR protein per g tissue in the absence of ryanodine, and 9.6 mg per g in its presence; calcium oxalate capacity gave similar numbers, 9.9 mg per g in the absence of ryanodine and 8.0 mg per g in its presence. The thapsigargin titration gave similar equivalent with or without ryanodine, indicating that the homogenate contained about 8.0 mg of SR per g tissue. Using 3H-ranodine binding as a marker, the cardiac content of SR was calculated to be 16.7 mg per g. These differences are attributed to the non-ideal behavior of these markers. Some of the Ca2+ uptake activity is not thapsigargin sensitive, and some of the 3H-ryanodine binding does not fractionate with the SR Ca2+ uptake activity.     

Image analysis of protein profiles from paired microvillous and basal syncytiotrophoblast plasma membranes from term human placenta and characterization of IgG binding to membrane vesicles

In contrast to the knowledge on the frequency and determinants of arterial diseases, little epidemiologic research has been carried out on venous diseases; this may be partly due to methodological problems in defining chronic venous insufficiency and in measuring these conditions with sufficient validity. Epidemiologic studies that were published after 1965 and that are not based on clinical series are reviewed; prevalence and incidence rates are reported. Studies of risk factors for varicose veins have largely resulted in inconsistent results; the sex difference is universal while the large geographical differences suggest strong environmental influences. For all other determinants much of the variation between studies is probably related to differences in definition, in population-sampling techniques, and in assessment methods. Several plausible etiologic theories on the causes and development of chronic venous insufficiency are supported or refuted by the epidemiologic studies. Further research is needed, whenever possible cross-cultural, with particular emphasis on clear definitions, valid methods, and a prospective study design.     

Energy-dependent uptake of calcium by the yeast Schizosaccharomyces pombe

1. In resting cells of the fission yeast Schizosaccharomyces pombe, the uptake of calcium is stimulated by the addition of 90 mM glucose in the presence as in the absence of respiration and inhibited by Antimycin A in the absence of exogenous carbon source. This uptake therefore requires fermentative or respiratory metabolic energy. 2. The calcium uptake by S. pombe exhibits saturation kinetics and high affinity for calcium. At external pH 4.5, the apparent Km is 45 muM ca2+ 400 muM of other divalent cations exert competitive inhibitions of calcium uptake in the following order of affinities: Sr2+ greater than Mn2+ greater than Co2+ greater than Mg2+. Inhibition by KCl is also observed but is of non-competitive type and requires high concentrations of the order of 40 mM. 3. At 30 degrees C, the uptake rate of calcium is about 10-times higher at pH 8925 than at pH 4.0. An extrusion of 45Ca2+, the rate of which is estimated to be lower than one-fifth of the uptake, is observed in the presence of glucose when the external pH is acid. 4. At external pH 4.5, low concentrations of lanthanum chloride, ruthenium red and hexamine cobaltichloride are inhibitory for the uptake of calcium by the yeast cells. 5. In presence of Antimycin A, the uncouplers: NaN3, dinitrophenol, and concentrations of crobonylcyanide m-chlorophenylhydrazone higher than 80 muM inhibit the calcium uptake by glycolysing cells. In the presence of glucose, the K+ ionophore Dio-9 dnhances severalfold the uptake of calcium even at 2 degrees C. 6. It is concluded that S. pombe possess an active transport system for low concentrations of calcium. This transport seems to be dependent on an electric potential (negative inside) across the cellular membrane.     

Oleic acid binding and transport capacity of human red cell membrane

Resealed human red cell membranes, ghosts, bind oleate (OL) by a limited number of sites when equilibrated at 37 degrees C, pH 7.3 with OL bound to bovine serum albumin (BSA) in molar ratios below 1.5. The binding capacity is 34 +/- 2.2 nmol g-1 ghosts with a dissociation equilibrium constant (37 degrees C) Kdm 1.38 +/- 0.15 fold Kd of albumin binding Kdm is temperature independent and approximately 7-8 nM. Exchange efflux kinetics at 0 degrees C to buffers of various albumin concentrations ([BSAy]) is biexponential and is analysed in terms of a three-compartment model. Accordingly the ratio of inner to outer membrane leaflet binding sites is 0.450 +/- 0.018 and the rate constant of unidirectional flux from inside to outside is 0.067 +/- 0.01 s-1. The rate constant of flux from the extracellular side of the membrane to BSAy increases with the square root of [BSAy] as expected of an unstirred layer effect. This provides an estimate of the dissociation rate constant of OL-BSA complex at 0 degrees C of 0.0063 +/- 0.0003 s-1. Exchange efflux from ghosts containing four different [BSAi] obeys the expected kinetics of a three-compartment approximation of the theoretical model. Accounting for the effect of an unstirred fluid inside ghosts, the rate coefficients fit the values predicted by the parameters obtained by the studies of albumin-free ghosts. The results show that the OL transport across the membrane is mediated exclusively by the asymmetrically distributed binding sites. The differences between transport sites of three long-chain fatty acids suggest that they are protein determined microdomains of phospholipids.     

Lack of effect of cocaine on lysine and alanine uptake in human placental villi or transfer in perfused human placenta

The effect of cocaine on lysine and alanine uptake in human placental villi and transfer across the dually perfused placenta was studied. Uptake (in terms of the intracellular to extracellular distribution ratio) of alanine and lysine was 2.81 +/- 0.30 (n = 5) and 1.45 +/- 0.24 (n = 5) respectively and was unaffected by cocaine (50-500 ng mL(-1) in the incubation medium. In the dually perfused placenta, the clearance index (ratio of amino acid to antipyrine clearance) was 0.35 +/- 0.03 and 0.30 +/- 0.05 and the transfer index (ratio of amino acid to L-glucose clearance) was 2.20 +/- 0.07 and 1.89 +/- 0.29 for lysine and alanine respectively. Cocaine at concentrations of 100 ng mL(-1) or 250 ng mL(-1) had no effect on the clearance of either amino acid. The results of this study indicate that concentrations of cocaine likely to be encountered in vivo do not affect uptake of lysine or alanine by placental villi or transfer across the perfused placental lobule, in contrast with the report that cocaine reduces uptake of alanine by placental vesicles. Experimental models must be critically evaluated before accepting the results as pertinent to a clinical situation.     

Expression of two alpha 2-adrenergic receptor subtypes in human placenta: evidence from direct binding studies

Adrenergic receptors may play an important role for mediating a variety of metabolic and haemodynamic effects of catecholamines including placental blood flow. The alpha-adrenergic receptors of the human placenta were characterized in vitro by the use of [3H]rauwolscine and [3H]prazosin as radioligands. Saturation experiments would suggest that the alpha-adrenoceptors in the human placenta are alpha 2. Comparative binding studies were performed, using recently synthesized compounds (Beecham Pharmaceuticals, UK) selective for alpha 2A (BRL-44408) and alpha 2B (BRL-41992) subtypes. The results indicate that human placenta contains at least two pharmacologically distinct alpha 2-adrenoceptor subtypes with approximately 60 per cent alpha 2A and 40 per cent alpha 2B receptors. In contrast with the pattern of increasing beta-adrenoceptor density, the concentration of alpha 2-adrenoceptors in term placentae is significantly lower than in placentae from the first trimester.     

Calcium uptake in preterminal central synapses: importance of mitochondria

Energy dependent 45Ca2+ uptake in the synaptosomal preparation from guinea pig cortex has been investigated. 45Ca2+ uptake was stimulated by ATP, the absolute value of uptake being dependent on the extent of synaptosomal disruption caused by osmotic shock. A quantitative comparison of microsomal and mitochondrial ATP-dependent 45Ca2+ uptake showed that only mitochondria had a large enough capacity to account for the Ca uptake levels observed in the synaptosomal preparation. ATP-stimulated 45Ca2+ uptake in mitochondria, 'intact' and 'shocked' synaptosomes was inhibited by atractyloside, DNP, oligomycin and ruthenium red but unaffected by antimycin A and rotenone. This was interpreted as evidence that mitochondria were responsible for ATP-dependent synaptosomal Ca2+ uptake, the increase in uptake seen on osmotic lysis being due to the deocclusion of intraterminal mitochondria. Synaptosomal and mitochondrial 45Ca2+ uptake was also stimulated by the mitochondrial respiratory substrate glutamate; this uptake was sensitive to antimycin A, DNP, rotenone and ruthenium red but insensitive to atractyloside or oligomycin thus indicating it was of mitochondrial origin. No change in glutamate-dependent 45Ca2+ uptake was seen on osmotic lysis of the synaptosomes as the expected increase due to the release of occluded mitochondria was counterbalanced by the damaging effect of hypo-osmotic shock on the glutamate-stimulated 45Ca2+ uptake process.     

Chloride channels in excised membrane patches from human platelets: effect of intracellular calcium

Interleukin-6 (IL-6) is one of the most important growth factors for myeloma cells. We examined the effect of recombinant IL-6 on the proliferation of five human myeloma cell lines, which were independently established AT Kawasaki Medical School. Only the KMS-11 cell line among these five lines showed growth enhancement induced by IL-6. Based on the results, a possible contribution of Ca(2+)-phospholipid-dependent protein kinase C (PKC) to the signal transduction in KMS-11 cells during growth enhancement was studied, since PKC may play an important role in malignant transformation or cell proliferation induced by some growth factors, such as IL-6. When exogenous IL-6 was added to KMS-11 culture, we observed (1) reduction of total PKC activity, and (2) translocation of PKC activity from its cytosol fraction to the membrane fraction. These findings may indicate that down regulation of PKC occurred during the myeloma cell proliferation induced by IL-6. However, IL-6 does not appear to be involved in cell proliferation and differentiation in the other cell lines studied.     

Esterified cholesterol accumulation induced by aggregated LDL uptake in human vascular smooth muscle cells is reduced by HMG-CoA reductase inhibitors

Vascular smooth muscle cell (VSMC) proliferation is a key event in the development of atherosclerotic lesions. VSMCs synthesize extracellular matrix, where low density lipoproteins (LDLs) are trapped and become aggregated (agLDL). The objective of this study was to investigate the cholesterol uptake and accumulation triggered by agLDL in comparison with native LDL (nLDL) on unstimulated and platelet-derived growth factor-stimulated human aortic VSMCs and the role of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on these processes. Esterified cholesterol (EC) accumulation induced by agLDL in VSMCs was correlated with the degree of aggregation and concentration. The EC content of VSMCs treated with 100 microg/mL of agLDL (80% aggregated) increased approximately 70-fold over that in VSMCs incubated with the same concentration of nLDL. Whereas nLDL-derived EC was increased approximately twofold in platelet-derived growth factor-stimulated VSMCs, there was no effect of platelet-derived growth factor (10(-9) mol/L) on the uptake of agLDL. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin (5 micromol/L) reduced EC accumulation derived from agLDL uptake by 58% and 35% in platelet-derived growth factor-stimulated and unstimulated VSMCs, respectively. This inhibition was overcome by geranylgeraniol (10 micromol/L) and partially by farnesol (10 micromol/L). Fluorescence microscopy of the cellular internalization of agLDL labeled with the fluorochrome 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine demonstrated that simvastatin reduces EC accumulation derived from agLDL by inhibiting its endocytosis and that the effect is completely reversed by geranygeraniol. These results indicate that agLDLs are rapidly internalized by human VSMCs and that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors modulate EC accumulation. These data suggest a possible mechanism by which statins could contribute to the passivation and stabilization of actively growing atherosclerotic lesions.