Functional differences in steroid sulfate uptake of organic anion transporter 4 (OAT4) and organic anion transporting polypeptide 2B1 (OATP2B1) in human placenta

Human trophoblasts depend on the supply of external precursors such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16alpha-OH-DHEA-S for synthesis of estrogens. Recently, we have characterized the uptake of DHEA-S by isolated mononucleated trophoblasts and identified different transporter polypeptides involved in this process. Immunohistochemistry of 1st and 3rd trimester placenta detected organic anion transporter 4 (OAT4) and organic anion transporting polypeptide 2B1 (OATP2B1, former name OATP-B) in cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast, indicating that both transporter polypeptides are involved in placental uptake of foetal derived steroid sulfates. In the present study we have characterized and compared the kinetics of DHEA-S and estrone sulfate (E(1)S) uptake by these transporters stably expressed in FlpIn -HEK293 cells using the Flp recombinase-mediated site-specific recombination. Uptake of E(1)S by OAT4- and OATP2B1-transfected cells was highly increased compared to the non-transfected cells. In contrast, DHEA-S uptake was only highly increased in OAT4 (40 times), but only weakly enhanced in OATP2B1 cells. The uptake of DHEA-S and E(1)S by OAT4 was partly Na(+)-dependent (about 50%), whereas uptake of DHEA-S by OATP2B1 was Na(+)-independent. Kinetic analysis of the initial uptake rates of E(1)S by OAT4 and OATP2B1 gave very similar values for K(m) (about 20microM) and V(max) (about 600pmol/(minxmg protein)). In contrast, the affinity of DHEA-S towards OATP2B1 was about 10 times lower (K(m)>200microM) then for OAT4 (K(m)=29microM). Our results suggest different physiological roles of the two transporter polypeptides in placental uptake of foetal derived steroid sulfates. OATP2B1 seems not to be involved in de novo synthesis of placental estrogens but may contribute to the clearance of estrogen sulfates from foetal circulation.     

Simultaneous determination of androstenediol 3-sulfate and dehydroepiandrosterone sulfate in human serum using isotope diluted liquid chromatography-electrospray ionization-mass spectrometry

A simple method for simultaneous determination of androstenediol 3-sulfate (Adiol-3S) and dehydroepiandrosterone sulfate (DHEA-S) in human serum using isotope diluted liquid chromatography-electrospray ionization-ion trap-mass spectrometry (LC-ESI-ion trap-MS) was developed. After addition of deuterated internal standards ([2H5]Adiol-3S and [2H4]DHEA-S), human serum (100 microl) was deproteinized with acetonitrile and then applied to a solid-phase extraction cartridge, Oasis HLB. The obtained steroid sulfates fraction was washed with hexane and then analyzed by LC-ESI-MS operated in the negative ion mode. The quantification ranges of Adiol-3S and DHEA-S were 10-400 ng/ml and 0.05-8 microg/ml, respectively. The method does not require the chemical or enzymatic hydrolysis of the conjugates and purification with high-performance liquid chromatography, and shows satisfactory reproducibility and accuracy. The concentrations of these sulfates in the sera of healthy male volunteers (n=14) were 19.2-245.3 mg/ml (Adiol-3S) and 0.175-5.16 microg/ml (DHEA-S), and those of patients with prostate cancer (n=19) were 15.3-182.7 ng/ml (Adiol-3S; four samples, not detectable) and 0.110-2.421 microg/ml (DHEA-S).     

Mechanism of n-butyrate uptake in the human proximal colonic basolateral membranes

Current studies were undertaken to characterize the mechanism of short-chain fatty acid (SCFA) transport in isolated human proximal colonic basolateral membrane vesicles (BLMV) utilizing a rapid-filtration n-[(14)C]butyrate uptake technique. Human colonic tissues were obtained from mucosal scrapings from organ donor proximal colons. Our results, consistent with the existence of a HCO(3)(-)/SCFA exchanger in these membranes, are summarized as follows: 1) n-[(14)C]butyrate influx was significantly stimulated into the vesicles in the presence of an outwardly directed HCO(3)(-) and an inwardly directed pH gradient; 2) n-[(14)C]butyrate uptake was markedly inhibited (approximately 40%) by anion exchange inhibitor niflumic acid (1 mM), but SITS and DIDS (5 mM) had no effect; 3) structural analogs e.g., acetate and propionate, significantly inhibited uptake of HCO(3)(-) and pH-gradient-driven n-[(14)C]butyrate; 4) n-[(14)C]butyrate uptake was saturable with a K(m) for butyrate of 17.5 +/- 4.5 mM and a V(max) of 20.9 +/- 1.2 nmol x mg protein(-1) x 5 s(-1); 5) n-[(14)C]butyrate influx into the vesicles demonstrated a transstimulation phenomenon; and 6) intravesicular or extravesicular Cl(-) did not alter the anion-stimulated n-[(14)C]butyrate uptake. Our results indicate the presence of a carrier-mediated HCO(3)(-)/SCFA exchanger on the human colonic basolateral membrane, which appears to be distinct from the previously described anion exchangers in the membranes of colonic epithelia.     

Progesterone Inhibits Folic Acid Transport in Human Trophoblasts

The aim of this work was to test the putative involvement of members of the ABC superfamily of transporters on folic acid (FA) cellular homeostasis in the human placenta. [(3)H]FA uptake and efflux in BeWo cells were unaffected or hardly affected by multidrug resistance 1 (MDR1) inhibition (with verapamil), multidrug resistance protein (MRP) inhibition (with probenecid) or breast cancer resistance protein (BCRP) inhibition (with fumitremorgin C). However, [(3)H]FA uptake and efflux were inhibited by progesterone (200 microM). An inhibitory effect of progesterone upon [(3)H]FA uptake and efflux was also observed in human cytotrophoblasts. Moreover, verapamil and ss-estradiol also reduced [(3)H]FA efflux in these cells. Inhibition of [(3)H]FA uptake in BeWo cells by progesterone seemed to be very specific since other tested steroids (beta-estradiol, corticosterone, testosterone, aldosterone, estrone and pregnanediol) were devoid of effect. However, efflux was also inhibited by beta-estradiol and corticosterone and stimulated by estrone. Moreover, the effect of progesterone upon the uptake of [(3)H]FA by BeWo cells was concentration-dependent (IC(50 )= 65 [range 9-448] microM) and seems to involve competitive inhibition. Also, progesterone (1-400 microM) did not affect either [(3)H]FA uptake or efflux at an external acidic pH. Finally, inhibition of [(3)H]FA uptake by progesterone was unaffected by either 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid (SITS), a known inhibitor of the reduced folate carrier (RFC), or an anti-RFC antibody. These results suggest that progesterone inhibits RFC. In conclusion, our results show that progesterone, a sterol produced by the placenta, inhibits both FA uptake and efflux in BeWo cells and primary cultured human trophoblasts.     

On the Mechanism of Ouabain-Induced Release of Acetylcholine from Synaptosomes

Ouabain (5 x 10(-8)-5 x 10(-4) M) was confirmed to cause a dose-dependent increase in [3H]acetylcholine ([3H]ACh) release, cytosolic free Ca2+ concentration ([Ca2+]i), and 22Na+ uptake in cerebrocortical synaptosomes of rats in the presence of extracellular Ca2+. Ouabain also caused a dose-dependent decrease in membrane potential. In a low-Na+ (10 mM) medium, ouabain failed to increase [3H]ACh release and [Ca2+]i. Tetrodotoxin (10(-6) M) had no effect on the ouabain-induced increase in both [3H]ACh release and [Ca2+]i but abolished the increase in 22Na+ uptake and partially inhibited the depolarizing effect. Verapamil (10(-6)-5 x 10(-4) M) inhibited the ouabain-induced increase in both [3H]ACh release and [Ca2+]i in a dose-dependent manner. Removal of extracellular Ca2+ abolished the effect of ouabain on [Ca2+]i but not on [3H]ACh release and 22Na+ uptake, regardless of the presence or absence of EGTA. In the absence of extracellular Ca2+, 10 mM Mg2+ blocked ouabain-induced [3H]ACh release, which was resistant to verapamil. These results suggest that ouabain can increase ACh release from synaptosomes without the preceding increases in intracellular Ca2+ and/or Na+ content. It seems likely that the removal of extracellular Ca2+ unmasks mechanisms of ouabain action different from those operating in the presence of Ca2+.     

The uptake of γ-aminobutyrate by organotypic cultures of chick spinal cord

1. Explants of spinal cord from 10-day chick embryos maintained for up to 16 days in culture rapidly accumulated gamma-amino[(3)H]butyrate when incubated at 25 degrees C or 36 degrees C in a medium containing 50nm-gamma-aminobutyrate. The mechanism of the uptake process has many of the properties of an active-transport system: it is Na(+)-dependent, temperature-sensitive, inhibited by ouabain, and displays saturation kinetics. The apparent K(m) for gamma-aminobutyrate is 1.7x10(-5)m, and V(max.) is 33pmol/min per g. 2. The rate of accumulation of gamma-amino[(3)H]butyrate in cultures between the ages of 3 and 16 days was remarkably constant and was not related to the morphological maturity of the spinal-cord explants. 3. The present demonstration in spinal-cord explants of an active transport system for gamma-aminobutyrate, already established for non-cultured nervous tissue, means that nervous-tissue culture can provide a convenient model for studying uptake processes in the central nervous system.     

Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2

We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) in a series of functional in vivo and in vitro studies. The apparent BBB efflux rate constant of [(3)H]DHEAS evaluated by the brain efflux index method was 2.68 x 10(-2) min(-1). DHEAS efflux transport was a saturable process with a Michaelis constant (K:(m)) of 32.6 microM: Significant amounts of [(3)H]DHEAS were determined in the jugular venous plasma by HPLC, providing direct evidence that most of the DHEAS is transported in intact form from brain to the circulating blood across the BBB. This efflux transport of [(3)H]DHEAS was significantly inhibited by common rat organic anion-transporting polypeptide (oatp) substrates such as taurocholate, cholate, sulfobromophthalein, and estrone-3-sulfate. Moreover, the apparent efflux clearance of [(3)H]DHEAS across the BBB (118 microl/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 microl/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [(3)H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K:(m) of 34.4 microM: and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [(3)H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [(3)H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB.     

In vivo and in vitro evidence for ACh-stimulated L-arginine uptake

Whereas L-arginine is clearly recognized as the precursor for nitric oxide synthesis, and its entry into endothelial cells via system y(+) transport is established, few data exist regarding the acute regulation of this transport process. We specifically investigated the effect of ACh and isoprenaline (Iso) on L-arginine uptake in the human forearm and in cultured bovine aortic endothelial cells (BAEC). Sixteen healthy males were studied. During a steady-state intra-arterial infusion of [(3)H]L-arginine (100 nCi/min), the effects of ACh (9.25 and 37 microg/min), Iso (25-50 and 200 microg/min), and sodium nitroprusside (SNP) (1-2 and 8 microg/min) on forearm plasma flow (FPF), L-[(3)H]arginine uptake, and L-[(3)H]citrulline release were determined. In parallel experiments, the effects of ACh, Iso, and SNP on L-[(3)H]arginine uptake were studied in BAEC. L-Arginine uptake was inversely related to FPF (r = -0.50; P < 0.005). At a similar FPF (ACh 56.82 +/- 9.25, Iso 58.49 +/- 5.56, SNP 57.92 +/- 4.96 ml/min; P = ns), intra-arterial ACh significantly increased forearm uptake of L-[(3)H]arginine (54,655 +/- 8,018 dpm/min), compared with that observed with either Iso (40,517.23 +/- 6,841 dpm/min; P = 0.01) or SNP (36,816 +/- 4,650 dpm/min; P = 0.011). This was associated with increased ACh-induced L-[(3)H]citrulline release compared with Iso and SNP (P = 0.046). Similarly, in BAEC, ACh significantly increased L-[(3)H]arginine uptake compared with control, Iso, or SNP (ACh 12.0 x 10(7) +/- 1.83 x 10(7) vs. control 6.67 x 10(7) +/- 1.16 x 10(7) vs. Iso 7.35 x 10(7) +/- 1.63 x 10(7) vs. SNP 6.01 x 10(7) +/- 1.11 x 10(7) fmol.min(-1).mg(-1) at 300 micromol/l L-arginine; P = 0.043). Taken together, these data indicate that ACh stimulates L-arginine uptake in cultured endothelial cells and in human forearm circulation, indicating the potential for acute modulation of endothelial L-arginine uptake.     

Role of lysosomal acid lipase in the intracellular metabolism of LDL-transported dehydroepiandrosterone-fatty acyl esters

Dehydroepiandrosterone-fatty acyl esters (DHEA-FAE) belong to a unique family of naturally occurring hydrophobic steroid hormone derivatives that are transported in circulating lipoproteins and may act as a source of dehydroepiendrosterone (DHEA) and other biologically active steroid hormones in cells. Here, we studied the metabolic fate of low-density lipoprotein-associated [(3)H]DHEA-FAE ([(3)H]DHEA-FAE-LDL) and the possible role of lysosomal acid lipase (LAL) in the hydrolysis of DHEA-FAE in cultured human cells. When HeLa cells were incubated with [(3)H]DHEA-FAE-LDL, the accumulation of label in the cellular fraction increased with incubation time and could be inhibited by excess unlabeled LDL, suggesting LDL receptor or LDL receptor-related receptor-dependent uptake. During 48 h of chase, decreasing amounts of [(3)H]DHEA-FAE were found in the cellular fraction, while in the medium increasing amounts of unesterified [(3)H]DHEA and its two metabolites, [(3)H]-5alpha-androstanedione (5alpha-adione) and [(3)H]androstenedione (4-adione), appeared. As LDL-cholesteryl ester hydrolysis is dependent on LAL activity, we depleted LAL from HeLa cells using small interfering RNAs and compared the hydrolysis of [(3)H]DHEA-FAE-LDL and [(3)H]cholesteryl-FAE-LDL. The results demonstrated a more modest but significant reducing effect on the hydrolysis of [(3)H]DHEA-FAE compared with [(3)H]cholesteryl-FAE. Moreover, experiments in LAL-deficient human fibroblasts (Wolman disease patient cells) showed that [(3)H]DHEA-FAE hydrolysis was not completely dependent on LAL activity. In summary, LDL-transported [(3)H]DHEA-FAE entered cells via LDL receptor or LDL receptor-related receptor-mediated uptake, followed by intracellular hydrolysis and further metabolism into 5alpha-adione and 4-adione that were excreted from cells. Although LAL contributed to the deesterification of DHEA-FAE, it was not solely responsible for the hydrolysis.     

Specificity of anion exchange mediated by mouse Slc26a6

Recently, CFEX, the mouse orthologue of human SLC26A6, was localized to the brush border membrane of proximal tubule cells and was demonstrated to mediate Cl(-)-formate exchange when expressed in Xenopus oocytes. The purpose of the present study was to examine whether mouse Slc26a6 can mediate one or more of the additional anion exchange processes observed to take place across the apical membrane of proximal tubule cells. Influx of [(14)C]formate into Slc26a6-expressing oocytes was inhibited by sulfate, oxalate, and p-aminohippurate (PAH), indicating affinity for these anions. Measurements of uptake of [(14)C]oxalate, [(14)C]PAH, and [(35)S]sulfate indicated that Slc26a6 can mediate transport of oxalate and sulfate but not PAH. Studies of the effect of external anions on [(14)C]oxalate efflux demonstrated Slc26a6-mediated Cl(-)-oxalate, oxalate-formate, oxalate-oxalate, and oxalate-sulfate exchange. Two-electrode voltage clamp measurements indicated that Slc26a6-mediated Cl(-)-oxalate exchange is electrogenic. Intracellular pH recordings demonstrated that Slc26a6 can mediate Cl(-)-HCO(3)(-) exchange, but Cl(-)-OH(-) exchange was not detected. The presence of 100 microm oxalate inhibited the rate of Cl(-)-HCO(3)(-) exchange by 60%. We conclude that mouse Slc26a6 has affinity for oxalate, sulfate, and HCO(3)(-) in addition to Cl(-) and formate and can function in multiple exchange modes involving pairs of these anions. In the presence of high oxalate concentrations as found in renal tubular fluid and urine, Slc26a6 may largely function as an electrogenic Cl(-)-oxalate exchanger.     

Alveolar epithelial barrier functions in ventilated perfused rabbit lungs

We employed ultrasonic nebulization for homogeneous alveolar tracer deposition into ventilated perfused rabbit lungs. (22)Na and (125)I-albumin transit kinetics were monitored on-line with gamma detectors placed around the lung and the perfusate reservoir. [(3)H]mannitol was measured by repetitive counting of perfusion fluid samples. Volume of the alveolar epithelial lining fluid was estimated with bronchoalveolar lavage with sodium-free isosmolar mannitol solutions. Sodium clearance rate was -2.2 +/- 0.3%/min. This rate was significantly reduced by preadministration of ouabain/amiloride and enhanced by pretreatment with aerosolized terbutaline. The (125)I-albumin clearance rate was -0.40 +/- 0.05%/min. The appearance of [(3)H]mannitol in the perfusate was not influenced by ouabain/amiloride or terbutaline but was markedly enhanced by pretreatment with aerosolized protamine. An epithelial lining fluid volume of 1.22 +/- 0.21 ml was calculated in control lungs. Fluid absorption rate was 1.23 microl x g lung weight(-1) x min(-1), which was blunted after pretreatment with ouabain/amiloride. We conclude that alveolar tracer loading by aerosolization is a feasible technique to assess alveolar epithelial barrier properties in aerated lungs. Data on active and passive sodium flux, paracellular solute transit, and net fluid absorption correspond well to those in previous studies in fluid-filled lungs; however, albumin clearance rates were markedly higher in the currently investigated aerated lungs.     

Metabolism of dehydroepiandrosterone sulfate and estrone-sulfate by human platelets

The aim of the present research was to study the uptake of DHEAS, and to establish the intracrine capacity of human platelets to produce sex steroid hormones. The DHEAS transport was evaluated through the uptake of [(3)H]-DHEAS in the presence or absence of different substrates through the organic anion transporting polypeptide (OATP) family. The activity of sulfatase enzyme was evaluated, and the metabolism of DHEAS was measured by the conversion of [(3)H]-DHEAS to [(3)H]-androstenedione, [(3)H]-testosterone, [(3)H]-estrone and [(3)H]-17beta-estradiol. Results indicated the existence in the plasma membrane of an OATP with high affinity for DHEAS and estrone sulphate (E(1)S). The platelets showed the capacity to convert DHEAS to active DHEA by the steroid-sulfatase activity. The cells resulted to be a potential site for androgens production, since they have the capacity to produce androstenedione and testosterone; in addition, they reduced [(3)H]-estrone to [(3)H]-17beta-estradiol. This is the first demonstration that human platelets are able to import DHEAS and E(1)S using the OATP family and to convert DHEAS to active DHEA, and to transform E(1)S to 17beta-estradiol.     

Characterization of an organic anion transport system in a placental cell line

Transporters within the placenta play a crucial role in the distribution of nutrients and xenobiotics across the maternal-fetal interface. An organic anion transport system was identified on the apical membrane of the rat placenta cell line HRP-1, a model for the placenta barrier. The apical uptake of 3H-labeled organic anion estrone sulfate in HRP-1 cells was saturable (Km = 4.67 microM), temperature and Na+ dependent, Li+ tolerant, and pH sensitive. The substrate specificity of the transport system includes various steroid sulfates, such as beta-estradiol 3,17-disulfate, 17 beta-estradiol 3-sulfate, and dehydroepiandrosterone 3-sulfate (DHEAS) but does not include taurocholate, p-aminohippuric acid (PAH), and tetraethylammonium. Preincubation of HRP-1 cells with 8-bromo-cAMP (a cAMP analog) and forskolin (an adenylyl cyclase activator) acutely stimulated the apical transport activity. This stimulation was further enhanced in the presence of IBMX (a phosphodiesterase inhibitor). Together these data show that the apical membrane of HRP-1 cells expresses an organic anion transport system that is regulated by cellular cAMP levels. This transport system appears to be different from the known taurocholate-transporting organic anion-transporting polypeptides and PAH-transporting organic anion transporters, both of which also mediate the transport of estrone sulfate and DHEAS.     

Effects of autonomic blockers on linear and nonlinear indexes of blood pressure and heart rate in SHR

Endothelium-derived hyperpolarizing factor (EDHF) is released in response to agonists such as ACh and bradykinin and regulates vascular smooth muscle tone. Several studies have indicated that ouabain blocks agonist-induced, endothelium-dependent hyperpolarization of smooth muscle. We have demonstrated that epoxyeicosatrienoic acids (EETs), cytochrome P-450 metabolites of arachidonic acid, function as EDHFs. To further test the hypothesis that EETs represent EDHFs, we have examined the effects of ouabain on the electrical and mechanical effects of 14,15- and 11,12-EET in bovine coronary arteries. These arteries are relaxed in a concentration-dependent manner to 14,15- and 11,12-EET (EC(50) = 6 x 10(-7) M), bradykinin (EC(50) = 1 x 10(-9) M), sodium nitroprusside (SNP; EC(50) = 2 x 10(-7) M), and bimakalim (BMK; EC(50) = 1 x 10(-7) M). 11,12-EET-induced relaxations were identical in vessels with and without an endothelium. Potassium chloride (1-15 x 10(-3) M) inhibited [(3)H]ouabain binding to smooth muscle cells but failed to relax the arteries. Ouabain (10(-5) to 10(-4) M) increased basal tone and inhibited the relaxations to bradykinin, 11,12-EET, and 14,15-EET, but not to SNP or BMK. Barium (3 x 10(-5) M) did not alter EET-induced relaxations and ouabain plus barium was similar to ouabain alone. Resting membrane potential (E(m)) of isolated smooth muscle cells was -50.2 +/- 0.5 mV. Ouabain (3 x 10(-5) and 1 x 10(-4) M) decreased E(m) (-48.4 +/- 0.2 mV), whereas 11,12-EET (10(-7) M) increased E(m) (-59.2 +/- 2.2 mV). Ouabain inhibited the 11,12-EET-induced increase in E(m). In cell-attached patch clamp studies, 11,12-EET significantly increased the open-state probability (NP(o)) of a calcium-activated potassium channel compared with control cells (0.26 +/- 0.06 vs. 0.02 +/- 0.01). Ouabain did not change NP(o) but blocked the 14,15-EET-induced increase in NP(o). These results indicate that: 1) EETs relax coronary arteries in an endothelium-independent manner, 2) unlike EETs, potassium chloride does not relax the coronary artery, and 3) ouabain inhibits bradykinin- and EET-induced relaxations as has been reported for EDHF. These findings provide further evidence that EETs are EDHFs.     

SP-A is necessary for increased clearance of alveolar DPPC with hyperventilation or secretagogues

The role of surfactant protein-A (SP-A) in pulmonary uptake and metabolism of [(3)H]dipalmitoylphosphatidylcholine ([(3)H]DPPC) was studied in SP-A gene-targeted mice (SP-A -/-). Unilamellar liposomes were instilled into the trachea of anesthetized mice. Uptake was measured as dpm in lungs plus liver and kidney for in vivo experiments and in lungs and perfusate for isolated lung experiments. [(3)H]DPPC uptake increased with CO(2)-induced hyperventilation in wild-type mice (SP-A +/+) but was unchanged in SP-A -/-. Secretagogue treatment approximately doubled the uptake of [(3)H]DPPC in isolated lungs from SP-A +/+ but had no effect in SP-A -/-. Lungs degraded 23 +/- 1.2% of internalized [(3)H]DPPC in SP-A +/+ and 36 +/- 0.6% in SP-A -/-; degradation increased with 8-bromoadenosine 3',5'-cyclic monophosphate in SP-A +/+ but was unchanged in SP-A -/-. Activity of lysosomal-type phospholipase A(2) (PLA(2)) was significantly greater in lungs from SP-A -/- compared with SP-A +/+. Thus SP-A is necessary for lungs to respond to hyperventilation or secretagogues with increased DPPC uptake and also modulates the PLA(2)-mediated degradation of internalized DPPC.     

Role of clathrin- and actin-dependent endocytotic pathways in lung phospholipid uptake

We evaluated the contribution of endocytotic pathways to pulmonary uptake of surfactant lipids from the alveolar space. Resting and stimulated 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) uptake of unilamellar liposomes labeled with either [(3)H]dipalmitoylphosphatidylcholine ([(3)H]DPPC) or 1-palmitoyl-2-[12-(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl-phosphatidylcholine (NBD-PC) was studied in isolated perfused rat lungs and isolated type II cells. Amantadine and phenylarsine oxide, inhibitors of clathrin-mediated endocytosis, each decreased [(3)H]DPPC uptake under resting conditions by approximately 40%; their combination had no additional effect. Cytochalasin D, an inhibitor of actin-dependent processes, reduced liposome uptake by 55% and potentiated the effect of either clathrin inhibitor alone. Relative inhibition for all agents was higher in the presence of 8-Br-cAMP. The effect of inhibitors was similar for liposomes labeled with [(3)H]DPPC or NBD-PC. By fluorescence microscopy, NBD-PC taken up by lungs was localized primarily to alveolar type II cells and was localized to lamellar bodies in both lungs and isolated cells. These studies indicate that both clathrin-mediated and actin-mediated pathways are responsible for endocytosis of DPPC-labeled liposomes by alveolar type II cells in the intact lung.     

Urotensin-II activates L-arginine/nitric oxide pathway in isolated rat aortic adventitia

Urotensin-II (U-II), a cyclic peptide widely expressed in blood vessels, has diverse vascular actions that range from potent vasoconstriction to vasodilation. Although, U-II-induced vasodilation has been shown to be partially dependent on nitric oxide (NO), the involvement of vascular adventitia-derived NO, remains unknown. The present study aimed to elucidate the activation of U-II on L-arginine/NO pathway in isolated rat aortic adventitia. In adventitia of thoracic and abdominal aortas, the l-arginine/NO pathway was similarly characterized: the uptake of l-[(3)H]arginine was Na(+)-independent, with the peak occurring over around 40 min incubation; the total NO synthase (NOS) activity was mostly calcium-independent (>90%), and significantly inhibited by a specific iNOS inhibitor AMT; the production of NO metabolites nitrate and nitrite (NO(x)) was stimulated by L-arginine but not by D-arginine. In aortic adventitia exposed to rat U-II (10(-9) and 10(-8)M) for 6 h, the V(max) of l-[(3)H]arginine uptake over 40 min incubation was significantly increased, while the K(m) of l-[(3)H]arginine uptake showed no significant change. Besides, the iNOS mRNA level was up-regulated, the total NOS activity, largely calcium-independent, was significantly induced, and the NO(x) production was significantly stimulated by U-II. According to the same protocol as U-II, the positive control lipopolysaccharide (LPS, 10 microg/ml), which had been established to activate adventitial L-arginine/NO pathway, increased l-[(3)H]arginine uptake, iNOS activity and NO(x) production to a greater extent than U-II. In addition, the total NOS activities induced by 3 and 6h incubation of U-II and LPS were significantly inhibited by a specific inhibitor of protein synthesis, actinomycin D. In conclusion, the results showed that rat U-II activated L-arginine/NOS/NO pathway in rat aortic adventitia, suggesting a potential contributive role of adventitia-derived NO in the vasodilator response of U-II.     

Identification and comparsion of bile acid-binding polypeptides in ileal basolateral membrane

Summary Bile acid-binding polypeptides were examined using basolateral membrane vesicles and enterocytes isolated from rat ileum. The uptake of a photolabile taurocholate derivative, (7,7,-azo-3, 12-dihydroxy-5[3-³H]cholan-24-oyl)-2-aminoethanesulfonate, 7,7-azo-TC, in ileal vesicles preloaded with paraaminohippurate (PAH) was stimulated with respect to uptake in unpreloaded vesicles. The PAH-transstimulated uptake of 7,7-azo-TC was inhibited by taurocholate and vice versa. Irradiation of membrane vesicles in the presence of 7,7-azo-TC irreversibly inhibited PAH-transtimulated taurocholate uptake. Photoaffinity labeling of basolateral membrane vesicles directly with [³H] 7,7-azo-TC and separation of proteins by SDS-PAGE revealed incorporation of radioactivity into several polypeptides. Photoaffinity labeling of vesicles in the presence of taurocholate inhibited the labeling of 54,000 and 59,000 mol. wt. polypeptides. The efflux of taurocholate from ileal enterocytes wascis-inhibited by 7,7-azo-TC andtransstimulated by PAH. Irradiation of enterocytes in the presence of 7,7-azo-TC inhibited taurocholate efflux greater than the presence of 7.7-azo-TC in the dark. When enterocytes that were irradiated in the presence of [³H] 7,7-azo-TC were fractionated and the resultant basolateral membrane fraction was subjected to SDS-PAGE, incorporation of radioactivity into the 54,000 and 59,000 mol. wt. polypeptides was seen. In contrast, when the brush-border membrane fraction was subjected to SDS-PAGE, greatest incorporation of radioactivity was seen in the previously described 99,000 mol. wt. polypeptide. These studies suggest that 7,7-azo-TC shared transporters with natural bile acid and identified polypeptides that may be involved in bile acid and identified polypeptides that may be involved in bile acid transport across the basolateral membrane and differ from that seen in the brush-border membrane of the ileal epithelial cell.     

Lack of effect of insulin or insulin-like growth factor I on the steroid sulfatase activity of human placental cytotrophoblasts

Hyperinsulinemia is known to reduce serum dehydroepiandrosterone sulfate (DHEA-S) levels in normal females. A possible mechanism for this phenomenon would be an insulin-mediated increase in steroid sulfatase activity, with insulin acting either via activation of the insulin receptor or via cross-reaction with the insulin-like growth factor I (IGF-I) receptor. Using a well characterized human cytotrophoblast system, the presence of steroid sulfatase activity in isolated cytotrophoblasts was documented. Half maximal cellular hydrolysis of DHEA-S was observed at a substrate concentration of 9.6-14.5 microM, and maximal hydrolysis at a concentration of 75-100 microM. The hypothesis that insulin increases steroid sulfatase activity was examined by exposing cytotrophoblasts to supraphysiological concentrations of either insulin (2 micrograms/ml) or IGF-I (20 ng/ml) for 24 h and then measuring the rate of DHEA-S hydrolysis. Insulin failed to affect cytotrophoblastic steroid sulfatase activity, irrespective of whether the substrate concentration was 20 microM or 100 microM. IGF-I also exerted no effect on steroid sulfatase activity. These data indicate that neither insulin nor IGF-I affect the steroid sulfatase activity of human cytotrophoblasts. An effect of insulin or IGF-I on the steroid-sulfatase activity of other tissues has not been excluded. These observations suggest that the decline in serum DHEA-S levels during hyperinsulinemia is not mediated via an insulin-induced increase in steroid sulfatase activity.     

Novel lipoidal derivatives of pregnenolone and dehydroepiandrosterone and absence of their sulfated counterparts in rodent brain

A new sample preparation method coupled to GC-MS analysis was developed and validated for quantification of sulfate esters of pregnenolone (PREG-S) and dehydroepiandrosterone (DHEA-S) in rat brain. Using a solid-phase extraction recycling protocol, the results show that little or no PREG-S and DHEA-S (<1 pmol/g) is present in rat and mouse brain. These data are in agreement with studies in which steroid sulfates were analyzed without deconjugation. We suggest that the discrepancies between analyses with and without deconjugation are caused by internal contamination of brain extract fractions, supposed to contain steroid sulfates, by lipoidal forms of PREG and DHEA (L-PREG and L-DHEA, respectively). These derivatives can be acylated very efficiently with heptafluorobutyric anhydride and triethylamine, and their levels in rodent brain (approximately 1 nmol/g) are much higher than those of their unconjugated counterparts. They are distinct from fatty acid esters, and preliminary data do not favor structures such as sulfolipids or sterol peroxides. Noncovalent interactions between steroids and proteolipidic elements, such as lipoproteins, could account for some experimental data. Given their abundance in rodent brain, the structural characterization and biological functions of L-PREG and L-DHEA in the central nervous system merit considerable attention.