Rapid sulfation of 3,3',5'-triiodothyronine in native Xenopus laevis oocytes

Sulfation is an important metabolic pathway facilitating the degradation of thyroid hormone by the type I iodothyronine deiodinase. Different human and rat tissues contain cytoplasmic sulfotransferases that show a substrate preference for 3,3'-diiodothyronine (3,3'-T2) > T3 > rT3 > T4. During investigation of the expression of plasma membrane transporters for thyroid hormone by injection of rat liver RNA in Xenopus laevis oocytes, we found uptake and metabolism of iodothyronines by native oocytes. Groups of 10 oocytes were incubated for 20 h at 18 C in 0.1 ml medium containing 500,000 cpm (1-5 nM) [125I]T4, [125I]T3, [125I]rT3, or [125I]3,3'-T2. In addition, cytosol prepared from oocytes was tested for iodothyronine sulfotransferase activity by incubation of 1 mg cytosolic protein/ml for 30 min at 21 C with 1 microM [125I]T4, [125I]T3, [125I]rT3, or [125I]3,3'-T2 and 50 microM 3'-phosphoadenosine-5'-phosphosulfate. Incubation media, oocyte extracts, and assay mixtures were analyzed by Sephadex LH-20 chromatography for production of conjugates and iodide. After 20-h incubation, the percentage of added radioactivity present as conjugates in the media and oocytes amounted to 0.9 +/- 0.2 and 1.0 +/- 0.1 for T4, less than 0.1 and less than 0.1 for T3, 32.5 +/- 0.4 and 29.3 +/- 0.2 for rT3, and 3.8 +/- 0.3 and 2.3 +/- 0.2 for 3,3'-T2, respectively (mean +/- SEM; n = 3). The conjugate produced from rT3 was identified as rT3 sulfate, as it was hydrolyzed by acid treatment. After injection of oocytes with copy RNA coding for rat type I iodothyronine deiodinase, we found an increase in iodide production from rT3 from 2.3% (water-injected oocytes) to 46.2% accompanied by a reciprocal decrease in rT3 sulfate accumulation from 53.7% to 7.1%. After 30-min incubation with cytosol and 3'-phosphoadenosine-5'-phosphosulfate, sulfate formation amounted to 1.8% for T4, less than 0.1% for T3, 77.9% for rT3, and 2.9% for 3,3'-T2. These results show that rT3 is rapidly metabolized in native oocytes by sulfation. The substrate preference of the sulfotransferase activity in oocytes is rT3 > 3,3'-T2 > T4 > T3. The physiological significance of the high activity for rT3 sulfation in X. laevis oocytes remains to be established.     

Hepatic sinusoidal membrane transport of anionic drugs mediated by anion transporter Npt1

The purpose of our study was to establish the localization of the anion transporter Npt1 in liver and the relevance of Npt1 to carrier-mediated hepatic transport of beta-lactam antibiotics. Immunocytochemical examination of mouse liver with antiserum for Npt1 showed basolateral (sinusoidal) membrane localization. Function of Npt1 was characterized in Xenopus laevis oocytes. Injection of in vitro-transcribed cRNA into oocytes resulted in an increased uptake of [14C]benzylpenicillin (PCG). The Npt1-mediated uptake was saturable with a Michaelis constant (Km) of 0.46 +/- 0.18 mM and a maximum rate (Vmax) of 46.6 +/- 8.5 pmol/60 min/oocyte, and the uptake of [14C]PCG was independent of Na+ and pH, but dependent on chloride ion. Npt1-mediated [14C]PCG uptake was inhibited by several beta-lactam antibiotics and probenecid. Oocytes injected with Npt1-cRNA demonstrated significantly enhanced transport activity for other anionic compounds such as [14C]faropenem, [14C]foscarnet and [3H]mevalonic acid, as well as [14C]PCG, compared with water-injected oocytes. In conclusion, Npt1 is suggested to participate in hepatic sinusoidal membrane transport of organic anions such as beta-lactam antibiotics as well as inorganic anions for the efflux from hepatocyte-to-blood direction.     

Expression of a human renal sodium nucleoside cotransporter in Xenopus laevis oocytes

In this study, Xenopus laevis oocytes injected with poly(A)+ RNA (mRNA) isolated from human kidney were used to express a Na(+)-nucleoside cotransporter. Na(+)-stimulated [3H]thymidine uptake was enhanced 2-3-fold in oocytes injected with 50 ng poly(A)+ RNA and 4-5-fold in oocytes injected with 20 ng of a size-fractionated human renal cortex mRNA fragment (2-3 kb) in comparison with water-injected oocytes. Na(+)-dependent thymidine uptake in oocytes injected with the 2-3 kb mRNA fragment was inhibited significantly by thymidine and guanosine but not by formycin B, consistent with the N4 Na(+)-nucleoside cotransporter. The Km (28 microM) of Na(+)-dependent thymidine uptake in the oocytes injected with the 2-3 kb mRNA fragment was similar to the Km (27 microM) of Na(+)-dependent thymidine uptake obtained in human renal brush border membrane vesicles. These data suggest for the first time that a Na(+)-nucleoside cotransporter from human kidney can be expressed in X. laevis oocytes.     

Identification of thyroid hormone transporters

Thyroid hormone action and metabolism are intracellular events that require transport of the hormone across the plasma membrane. We tested the possible involvement of the Na+/taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide (oatp1) in the hepatic uptake of the prohormone T4, the active hormone T3, and the metabolites rT3 and 3,3'-T2. Xenopus laevis oocytes were injected with 2.3 ng Ntcp or oatp1 cRNA and, after 2-3 days, incubated for 1 h at 25 degrees C with usually 0.1 microM 125I-labeled ligand. Uninjected oocytes showed marked uptake of iodothyronines and this was further increased by Ntcp and oatp1 cRNA, i.e., 1.9- and 2.8-fold for T4, 1.7- and 1.7-fold for T3, 1.8- and 6.0-fold for rT3, and 1.3- and 1.4-fold for 3,3'-T2, respectively. Mostly due to much lower uptake by uninjected oocytes, Ntcp and oatp1 cRNA induced larger, 12- to 76-fold increases in uptake of iodothyronine sulfates. The Ntcp cRNA-induced iodothyronine uptake was completely inhibited in Na+-deplete medium, whereas the oatp1 cRNA-induced uptake was not affected. These results suggest that hepatic uptake of thyroid hormones and their metabolites is mediated at least in part by Ntcp and oatp1.     

cAMP-activation of amiloride-sensitive Na+ channels from guinea-pig colon expressed in Xenopus oocytes

Guinea-pig distal colonic mRNA injection into Xenopus laevis oocytes resulted in expression of functional active epithelial Na+ channels in the oocyte plasma membrane. Poly(A)+ RNA was extracted from distal colonic mucosa of animals fed either a high-salt (HS) or a low-salt (LS) diet. The electrophysiological properties of the expressed amiloride-sensitive Na+ conductances were investigated by conventional two-electrode voltage-clamp and patch-clamp measurements. Injection of poly(A)+ RNA from HS-fed animals [from hereon referred to as HS-poly(A)+ RNA] into oocytes induced the expression of amiloride-sensitive Na+ conductances. On the other hand, oocytes injected with poly(A)+ RNA from LS-fed animals [LS-poly(A)+ RNA] expressed a markedly larger amount of amiloride-blockable Na+ conductances. LS-poly(A)+ RNA-induced conductances were completely inhibitable by amiloride with a Ki of 77 nM, and were also blocked by benzamil with a Ki of 1.8 nM. 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA), even in high doses (25 "mu"M), had no detectable effect on the Na+ conductances. Expressed amiloride-sensitive Na+ channels could be further activated by cAMP leading to nearly doubled clamp currents. When Na+ was replaced by K+, amiloride (1 "mu"M) showed no effect on the clamp current. Single-channel analysis revealed slow gating behaviour, open probabilities (Po) between 0.4 and 0.9, and slope conductances of 3. 8 pS for Na+ and 5.6 pS for Li+. The expressed channels showed to be highly selective for Na+ over K+ with a permeability ratio PNa/PK > 20. Amiloride (500 nM) reduced channel Po to values < 0.05. All these features make the guinea-pig distal colon of LS-fed animals an interesting mRNA source for the expression of highly amiloride-sensitive Na+ channels in Xenopus oocytes, which could provide new insights in the regulatory mechanism of these channels.     

The predominant contribution of oligopeptide transporter PepT1 to intestinal absorption of beta-lactam antibiotics in the rat small intestine

Although recent evidence suggests that certain beta-lactam antibiotics are absorbed via a specific transport mechanism, its nature is unclear. To confirm whether peptide transport in the rat can be largely ascribed to the intestinal oligopeptide transporter PepT1, the transporter has been functionally characterized and its significance in the intestinal absorption of beta-lactam antibiotics was evaluated. For evaluation of transport activity complementary RNA (cRNA) of rat PepT1 was synthesized in-vitro and expressed in Xenopus laevis oocytes. cRNA induced uptake of several beta-lactam antibiotics and the dipeptide [14C]glycylsarcosine; this was specifically inhibited by various dipeptides and tripeptides but not by their constituent amino acids or by tetra- or pentapeptides. The transport activity of PepT1 for beta-lactam antibiotics correlated well with their in-vivo intestinal transport and absorption. Furthermore, mutual inhibitory effects on uptake were observed between glyclsarcosine and beta-lactam antibiotics. Hybrid depletion of the functional expression of rat PepT1 in oocytes injected with rat intestinal epithelial total mRNA was studied using an antisense oligonucleotide corresponding to the 5'-coding region of PepT1. In oocytes injected with rat mRNA pre-hybridized with the antisense oligonucleotide against rat PepT1, the uptake of [14C]glycylsarcosine was almost completely abolished, whereas its uptake was not influenced by a sense oligonucleotide for the same region of PepT1. Similarly, the uptake of beta-lactam antibiotics was also reduced by the antisense oligonucleotide against rat PepT1. These results demonstrate that the intestinal proton-coupled oligopeptide transporter PepT1 plays a predominant role in the carrier-mediated intestinal absorption of beta-lactam antibiotics and native oligopeptides in the rat.     

Kappa-opioid receptors couple to inwardly rectifying potassium channels when coexpressed by Xenopus oocytes

Xenopus oocytes expressed kappa-opioid specific binding sites after injection of cRNA prepared from a clone of the rat kappa-opioid receptor. Coinjection of kappa receptor cRNA with cRNA coding for a G protein-linked, inwardly rectifying, K+ channel (GIRK1, or KGA) resulted in oocytes that responded to the kappa agonist U-69593 by activating a large (1.0-1.5-microA) K+ current. U-69593 exhibited an EC50 of 260 +/- 50 nM and was blocked by the opioid antagonists norbinaltorphimine and naloxone. The kappa agonist bremazocine was 200-fold more potent than U-69593 in eliciting K+ current but exhibited a partial agonist profile in this expression system. The present results indicate that stimulation of inwardly rectifying K+ channels may be a potential effector mechanism for kappa-opioid receptors.     

Membrane-bound calmodulin in Xenopus laevis oocytes as a novel binding site for melatonin

Melatonin has been suggested as a physiological antagonist of calmodulin. In this work, we have characterized melatonin binding sites in Xenopus laevis oocyte membranes. Binding of [125I]melatonin by X. laevis oocyte membranes fulfills all criteria for binding to a receptor site. Binding was dependent on time, temperature, and membrane concentration and was stable, reversible, saturable, and specific. The binding site was also pharmacologically characterized. Stoichiometric studies showed a high-affinity binding site with a Kd of 1.18 nM. These data are in close agreement with data obtained from kinetic studies (Kd=0.12 nM). In competition studies, we observed a low-affinity binding site (Kd=63.41 microM). Moreover, the binding site was characterized as calmodulin. Thus, binding was dependent on calcium and blocked by anti-CaM antibodies in a concentration-dependent manner. Calmodulin inhibitor chlorpromazine also inhibited binding of the tracer. From these results, it is suggested that membrane-bound calmodulin acts as a melatonin binding site in Xenopus laevis oocytes, where it might couple cellular activities to rhythmic circulating levels of melatonin. This hypothesis correlates with the previous findings describing melatonin as a physiological antagonist of calmodulin.     

Leucine transport in Xenopus laevis oocytes: functional and morphological analysis of different defolliculation procedures

L-leucine uptake in stage V Xenopus laevis oocytes was affected by the specific methods used to remove the follicle cells. In the presence of 100 mM NaCl, L-leucine uptake was reduced by 67.5% +/- 5.7 when defolliculation was performed enzymatically by collagenase treatment, whereas the reduction was 30.5% +/- 6.4 after mechanical defolliculation. The Na(+)-dependent uptake of 0.1 mM L-leucine was 18.6 +/- 4.6 pmol oocyte-1 40 min-1 in folliculated oocytes and 5.6 +/- 1.9 in collagenase defolliculated oocytes (means +/- SE). L-leucine uptake was not affected by the removal of the follicular layer if defolliculation occurred after the transport period; radiolabeled L-leucine is therefore not taken up into a compartment that is removed by the defolliculation process. The different L-leucine uptake rates observed in folliculated and defolliculated oocytes were not due to non-specific L-leucine binding to membranes. L-leucine kinetics showed that the L-leucine Vmax and Km values were lower in oocytes deprived of the follicular layer than in control oocytes enveloped in intact follicular layers. The Vmax and Km values of Na(+)-dependent L-leucine transport, calculated from data obtained the day after defolliculation by collagenase treatment, were: 16 +/- 1.5 pmol oocyte-1 40 min-1 and 57 +/- 21 mumol (mean +/- SD). The Na(+)-activation curve of 0.1 mM L-leucine was hyperbolic in folliculated oocytes and sigmoidal in defolliculated oocytes. The morphological analysis performed in parallel with the transport experiments showed that after defolliculation, the fibers forming the vitelline membrane tended to be arranged in a more regular orthogonal array, and the number of oocyte microvilli was reduced after collagenase treatment. Mechanical defolliculation did not appreciably affect the oocyte microvilli, however this procedure did not completely remove all follicle cells. The damage to collagenase treated oocytes was reversible, and the functional and structural features of most oocytes improved upon subsequent in vitro incubation. The recovery process seemed to involve protein synthesis in view of the increased value of L-leucine Vmax, and microscopic observation showing recovery of the microvillar apparatus.     

Functional expression of the parathyroid cell calcium receptor in Xenopus oocytes

Various studies suggest the existence of a plasma membrane receptor on parathyroid cells that senses changes in the concentration of extracellular Ca2+. To test this hypothesis, Xenopus laevis oocytes were injected with poly(A)(+)-enriched mRNA from bovine parathyroid cells and examined for their ability to respond to increases in the concentration of extracellular Ca2+ or other polycations. Cytosolic Ca2+ concentrations were measured indirectly by recording Cl- currents through the endogenous, cytosolic Ca(2+)-activated Cl- channel. Increasing the concentration of extracellular Ca2+ (from 0.7 to 5 mM) or Mg2+ (from 0.8 to 10 mM) elicited oscillatory increases in the Cl- current. Responses to either divalent cation were not observed in oocytes injected with water or with mRNA prepared from HL-60 cells or rat liver. Responses elicited by extracellular Mg2+ persisted when extracellular Ca2+ was reduced to low micromolar levels. La3+, Gd3+, or neomycin B also evoked oscillatory increases in the Cl- current in oocytes under conditions of low extracellular Ca2+ levels. These extracellular polycations all cause the mobilization of intracellular Ca2+ in oocytes injected with parathyroid cell mRNA like they do in intact parathyroid cells. The injection of parathyroid cell mRNA thus confers on oocytes the ability to detect and respond to changes in the concentration of extracellular polycations. The data provide compelling evidence for the existence of a cell surface Ca2+ receptor protein(s) on parathyroid cells that regulates cellular function.     

Ontogeny of iodothyronine deiodinases in human liver

The role of the deiodinases D1, D2, and D3 in the tissue-specific and time-dependent regulation of thyroid hormone bioactivity during fetal development has been investigated in animals but little is known about the ontogeny of these enzymes in humans. We analyzed D1, D2, and D3 activities in liver microsomes from 10 fetuses of 15-20 weeks gestation and from 8 apparently healthy adult tissue transplant donors, and in liver homogenates from 2 fetuses (20 weeks gestation), 5 preterm infants (27-32 weeks gestation), and 13 term infants who survived up to 39 weeks postnatally. D1 activity was determined using 1 microM [3',5'-125I]rT3 as substrate and 10 mM dithiothreitol (DTT) as cofactor, D2 activity using 1 nM [3',5'-125I]T4 and 25 mM DTT in the presence of 1 mM 6-propyl-2-thiouracil (to block D1 activity) and 1 microM T3 (to block D3 activity), and D3 activity using 10 nM [3,5-125I]T3 and 50 mM DTT, by quantitation of the release of 125I. The assays were validated by high performance liquid chromatography of the products, and kinetic analysis [Michaelis-Menten constant (Km) of rT3 for D1: 0.5 microM; Km of T3 for D3: 2 nM]. In liver homogenates, D1 activity was not correlated with age, whereas D3 activity showed a strong negative correlation with age (r -0.84), with high D3 activities in preterm infants and (except in 1 infant of 35 weeks) absent D3 activity in full-term infants. In microsomes, D1 activities amounted to 4.3-60 pmol/min/mg protein in fetal livers and to 170-313 pmol/min/mg protein in adult livers, whereas microsomal D3 activities were 0.15-1.45 pmol/min/mg protein in fetuses and <0.1 pmol/min/mg protein in all but one adult. In the latter sample, D3 activity amounted to 0.36 pmol/min/mg protein. D2 activity was negligible in both fetal and adult livers. These findings indicate high D1 and D3 activities in fetal human liver, and high D1 and mostly absent D3 activities in adult human liver. Therefore, the low serum T3 levels in the human fetus appear to be caused by high hepatic (and placental) D3 activity rather than caused by low hepatic D1 activity. The occasional expression of D3 in adult human liver is intriguing and deserves further investigation.     

Synthesis and radiolabeling of (S)-4-amino-5-iodo-2-methoxy-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide, the active enantiomer of [125I]iodozacopride, and re-evaluation of its 5-HT3 receptor affinity

We report an improved synthesis of unlabeled (S)-iodozacopride, the radiolabeling of (S)-[125I]iodozacopride via deschloro-(S)-zacopride, and a re-evaluation of its affinity for the 5-HT3 receptor. Unlabeled (S)-iodozacopride was prepared in seven steps from 4-aminosalicylic acid via alkaline hydrolysis of its 4-acetamide derivative. Catalytic hydrogenation of (S)-iodozacopride gave deschloro-(S)-zacopride, identical to that obtained from (S)-3-amino-quinuclidine and 4-amino-2-methoxybenzoic acid via its corresponding 1-imidazole derivative. Radioiodination to produce (S)-[125I]iodozacopride was accomplished by treatment of deschloro-(S)-zacopride with 5 mCi sodium 125iodide and chloramine-T in hydrochloric acid. Purification of the reaction products using an HPLC system capable of detecting chlorinated side-products revealed a mixture of 2.1 mCi (1.3 nmol) (S)-[125I]iodozacopride and (S)-zacopride (1.5 nmol). Saturation analysis of the binding of the purified (S)-[125I]iodozacopride to whole rat brain homogenates gave an estimated KD of 1.10 +/- 0.07 nM. As anticipated, this is approximately half the KD reported for binding of racemic [125I]iodozacopride, and differs from the previously reported value by an order of magnitude. Analysis of the apparent binding affinity of a 1:1 mixture of (S)-[125I]iodozacopride and (S)-zacopride suggests that the previous result may have been confounded by contamination of the product with unlabeled (S)-zacopride. Competition analysis of the displacement of (S)-[125I]iodozacopride binding by unlabeled (S)-iodozacopride and (S)-zacopride gave Ki values of 0.95 and 0.21 nM, respectively.     

Effect of extracellular pH on the myo-inositol transporter SMIT expressed in Xenopus oocytes

The myo-inositol transporter SMIT is expressed particularly at high extracellular osmolarity and serves to accumulate the osmolyte myo-inositol. Transport of myo-inositol is coupled to the cotransport of Na+ and is electrogenic. In Xenopus oocytes injected with mRNA encoding SMIT but not in water-injected oocytes, myo-inositol creates an inward current that is dependent on the ambient Na+ concentration. The present study has been performed to elucidate the pH dependence of myo-inositol-induced currents. Therefore, Xenopus oocytes were injected with mRNA encoding SMIT and two-electrode voltage-clamp studies were performed. The myo-inositol-induced currents in oocytes expressing SMIT were found to have a sigmoidal dependence on the ambient pH between pH 5.5 and 8.5 with an apparent Ki of 0.21+/-001 microM H+ and a Hill coefficient of 1.80+/-0.16. Kinetic analysis of the myo-inositol-induced currents at pH 8.0 and -90 mV holding potential revealed a Hill coefficient of 0.93+/-0.07 and an apparent Km for myo-inositol of 0.031+/-0.003 mM as well as a Hill coefficient of 1. 64+/-0.24 and an apparent Km of 38.8+/-4.1 mM for Na+. A decrease of the Na+ concentra-tion from 150 mM to 50 mM significantly altered the maximal observed current and increased the apparent Km for myo-inositol. Acidification to pH 6.5 significantly increased the apparent Km for myo-inositol and for Na+ to 0.057+/-0.005 mM and 73. 9+/-4.8 mM, respectively. The Hill coefficients for myo-inositol and Na+ were not affected and remained close to 1 for myo-inositol and 2 for Na+. In summary, acidification impedes SMIT-mediated myo-inositol transport at least partially by decreasing the affinity of the carrier for Na+. The impaired Na+ binding subsequently decreases binding and transport of myo-inositol.     

On the existence of polyadenylated histone mRNA in Xenopus laevis oocytes

In a variety of systems, histone mRNA has been shown to lack poly(A) (Adesnik and Darnell, 1972; Grunstein et al., 1973). We have found, however, that in Xenopus laevis oocytes, poly (A)-containing mRNA codes for histones, in a wheat germ cell-free system, based on the following criteria: first, co-migration with authentic X. laevis oocyte histones on polyacrylamide gels; second, no detectable incorporation of tryptophan; third, differential incorporation of lysine and methionine into histone fraction H2A; fourth, resistance of histone fraction H2A to cleavage with cyanogen bromide; and fifth, correspondence of tryptic peptide maps of partially purified cell-free products with authentic X. laevis oocyte histone. RNA which directs the synthesis of histones in the cell-free system is retained on oligo(dT)-cellulose, even after denaturation in 80% DMSO at 70 degrees C, thereby demonstrating the covalent attachment of polyadenylic acid sequences to the mRNA. Poly (A)- RNA (7S-14S fraction) was also found to code for histones using the same criteria. We discuss the significance of the finding that X. laevis oocytes contain two classes of histone mRNA as well as the potential developmental implications of this observation.     

Effect of carboxy-PTIO, a nitric oxide scavenger, on the proliferation of murine hematopoietic progenitor cells in vitro

Maitotoxin (MTX) may exert its toxic effect by activating ion conductances and has been shown to elicit a fertilization-like response in Xenopus laevis oocytes. In the present study we investigated the electrophysiological response of stage V-VI Xenopus oocytes to MTX using the two-microelectrode voltage-clamp technique. Membrane voltage (Vm) measurements demonstrated that MTX (50 pM to 1 nM) depolarized the oocytes from -49+/-7 to -14+/-1 mV. Subsequent replacement of bath Na+ by the impermeant cation NMDG (N-methyl-d-glucamine) shifted Vm from -14+/-1 to -53+/-5 mV (n=29). This indicates that MTX activates a cation conductance. Indeed, current measurements at a holding potential of -60 or -100 mV showed that within 10 s of MTX application an inward current component developed which was largely abolished by extracellular Na+ removal. After a 1-min application of 1 nM MTX the NMDG-sensitive current increased more than 100-fold from 0.14+/-0.03 microA to a peak value of 21+/-3 microA (n=11). The effect of MTX was concentration dependent with an EC50 of about 250 pM but only slowly reversible. Ion substitution experiments indicated that the stimulated conductance was nonselective for monovalent cations with a slight preference for NH4+ (2.1) > K+ (1.5) > Na+ (1.0) > Li+ (0.7). Regarding divalent cations, a complex biphasic response to extracellular Na+ replacement by Ca2+ was observed, which suggests that the stimulated channels may have a small Ca2+ permeability but that exposure to high extracellular Ca2+ enhances recovery from MTX stimulation. No significant conductance for Mn2+ was observed. Application of 1 mM benzamil, 1 mM amiloride, or 100 microM 1-(beta-[3-(4-Methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazol e hydrochloride (SK&F 96365) reduced the MTX-stimulated inward current by 81%, 62%, or 65%, respectively. Gd3+ had an inhibitory effect of 29% and 38% at concentrations of 10 microM or 100 microM, respectively. Flufenamic acid, niflumic acid, (RS)-(3,4-dihydro-6, 7-dimethoxyisoquinoline-1-gamma1)-2-phenyl-N,N-di-[2-(2,3, 4-trimethoxyphenyl)-ethyl]-acetamide (LOE908), and 3', 5'-dichlorodiphenylamine-2-carboxylic acid (DCDPC), known blockers of other nonselective cation channels, had no significant effect. We conclude that MTX activates a nonselective cation conductance in Xenopus oocytes. The underlying channels may be involved in changes in Vm that occur during the early stages of fertilization.     

Characterization of (S)-des-4-amino-3-[125I]iodozacopride ([125I]DAIZAC), a selective high-affinity radioligand for 5-hydroxytryptamine3 receptors

The 5-hydroxytryptamine(HT)3 receptor subtype is present in the central nervous system (CNS) in low abundance, and few selective radiolabeled antagonists with high specific activity are available to study these sites. DAIZAC [desamino-3-iodo-(S)-zacopride; (S)-5-chloro-3-iodo-2-methoxy-N-(1-azobicyclo-[2.2. 2]oct-3-yl)benzamide] is a compound with high affinity and selectivity for the 5-HT3 receptor. Scatchard analysis of specific binding to NCB-20 cell membranes gave a Bmax of 340 +/- 58 fmol/mg protein and a KD of 0.14 +/- 0.03 nM, which is in agreement with the value previously reported in rat brain (KD = 0.15 nM). Nonspecific binding of [125I]DAIZAC in NCB-20 cells was <1% of total binding at the KD for DAIZAC compared with 17% in the rat brain preparation. Unlabeled DAIZAC (10 microM) showed minimal ability to displace binding of radiolabeled ligands selected for their affinities for other CNS receptor and uptake carrier binding sites. The discrimination ratio of DAIZAC for the 5-HT3 receptor over the M1 muscarinic binding site, the non-5-HT3 site at which it was most potent, was >2800. Serotonergic antagonists at every other known CNS serotonergic binding sites (3-30 microM) were ineffective in displacing [125I]DAIZAC binding in rat brain membranes. Similarly, antagonists (3-30 microM) for other nonserotonergic receptors and uptake sites were ineffective in displacing [125I]DAIZAC binding. Autoradiographic studies showed highest specific binding in area postrema and nucleus solitarius, with intermediate levels of binding in entorhinal cortex and hippocampus. DAIZAC inhibited 5-HT3 receptor-mediated inward cation current in NCB-20 cells with an IC50 of 0.24 nM. [125I]DAIZAC is a potent and highly selective ligand for in vitro studies of the 5-HT3 receptor.     

Angiotensin I modulates Ca-transport systems in the rat heart through angiotensin II

The objective of this study was to determine the effect of angiotensin I (Ang I) treatment in vivo on two major Ca-transport systems-the L-type voltage dependent calcium channel (L-VDCC) and the Na/Ca exchanger in rat heart. For our experiments we used four groups of rats, treated differently with saline, Ang I, the ACE inhibitor enalapril and/or combination of both for 6 days, every 24 h. We observed an increase in the activity, and also in mRNA expression of the Na/Ca exchanger, after repeated administration of Ang I in vivo. The maximal binding capacity of Ca-antagonist PN 200-110, which binds to the alpha 1 subunit of the L-VDCC was elevated from 0.8-1.85 pg/mg protein. mRNA expression of the voltage-dependent calcium channels of L-type system was also upregulated by Ang I administration, but not when enalapril was applied simultaneously with Ang I. These results demonstrate that in vivo application of the Ang I significantly modulates not only the activity, but also expression of the Na/Ca exchanger and the L-VDCC in rat hearts through angiotensin II (Ang II). Since in the in vitro experiments on the isolated cardiomyocytes, Ang II (100 nM) increased the calcium uptake after depolarization, and the AT1 receptor agonist losartan prevented this increase, we assume that this regulation might involve the AT1 receptors.