Beef heart mitochondrial F1-ATPase: inhibition by azidoadenyl-5'-yl imidodiphosphates and cooperative binding of substrate.

Two ATP analogs, 2- and 8-azidoadenyl-5'-yl imidodiphosphate, were synthesized, purified and utilized as inhibitors of soluble beef heart mitochondrial F1-ATPase under non-photolytical conditions. In the range of 5 microM to 3 mM ATP, the initial rates of ATP hydrolysis in the presence and absence of the inhibiting ATP analogs can be adequately described by two pairs of Km and Vmax values (3 microM, 8.5 mumol ATP/min per mg; 255 microM, 42.0 mumol ATP/min per mg). With increasing inhibitor concentrations, the apparent Km,2 increases as in competitive inhibition, while Vmax,1 decreases as in non-competitive inhibition. The Ki values derived for both types of inhibition are similar, but strongly different for 2- and 8-azido-AMP-PNP (4 microM and 460 microM, respectively). The decrease of the high-affinity Vmax is compensated by an increase in low-affinity catalysis, resulting in a constant sum of maximal velocities. These data can be described by a model where two sites interact with negative cooperativity in binding of substrate.

Molecular cloning and functional characterization of two alternatively spliced Ntcp isoforms from mouse liver1.

To isolate the murine Na+/taurocholate cotransporting polypeptide (Ntcp), we screened a mouse liver cDNA library and identified Ntcp1, encoding a 362 amino acid protein and Ntcp2, encoding a 317 amino acid protein which had a shorter C-terminal end. Both isoforms mediated saturable Na+-dependent transport of taurocholate when expressed in Xenopus laevis oocytes. Analysis of the gene revealed that Ntcp2 is produced by alternative splicing where the last intron is retained.

Functional expression of the rat liver canalicular isoform of the multidrug resistance-associated protein.

The rat hepatocanalicular isoform (called mrp2) of the human multidrug resistance-associated protein (MRP) has been cloned and transiently expressed in COS-7 cells and in Xenopus laevis oocytes. In both systems mrp2 expression induced a markedly increased efflux of intracellularly formed [14C]2,4-dinitrophenyl-S-glutathione. Injection of mrp2 cRNA into oocytes also stimulated efflux of [3H(N)]leukotriene C4. Furthermore, mrp2 mRNA was markedly decreased in the liver of the transport mutant TR rat, which has a congenital defect in the biliary excretion of glutathione-S conjugates and of other divalent organic anions. The study provides a direct demonstration of mrp2-mediated transport function and supports the concept that mrp2 represents the canalicular multispecific organic anion transporter (cMOAT) of mammalian liver.

First-pass elimination of a peptidomimetic thrombin inhibitor is due to carrier-mediated uptake by the liver. Interaction with bile acid transport systems.

CRC 220 (4-methoxy-2, 3, 6-trimethylphenylsulfonyl-L-aspartyl-D-4-amidinophenylalanyl -piperidide) is a competitive peptide-based trombin inhibitor with high affinity to human alpha-thrombin (Ki 2.5 nM). The amphiphilic compound exhibits virtually no systemic bioavailability despite proteolytic stability and proven enteral absorption. After intravenous application (V. jejunalis) in rats CRC 220 is almost completely excreted into bile. Simultaneous administration of bile acids considerably decreases this first-pass elimination. CRC 220 is extensively taken up in isolated rat hepatocytes by a saturable carrier-mediated transport with Km 23.7 microM and Vmax 775 pmol x mg-1 x min-1. A large part of this transport is energy-dependent. At temperatures above 20 degrees C, the uptake is accelerated exponentially. The activation energy amounts to 82 kj/mol. A minor portion of CRC 220 uptake occurs by physical diffusion with a permeability coefficient of 7.83 x 10(-7) cm/sec at 12 degrees C. Sodium ions energize CRC 220 uptake. Replacement of sodium by choline or lithium decreases the transport rate of 23-40%. In addition, a negative membrane potential facilitates the uptake. CRC 220 transport is only observed in hepatocytes: it is absent in BHK, FAO, HepG2, HPCT 1E3, and HPCT 1E3-TC cells. In the presence of 4-amidinophenylalanine derivatives, CRC 220 uptake is considerably decreased. Inhibition also occurs with bile acids and bromosulfophthalein, but less with bumetanide. Because CRC 220 inhibits bile acid uptake into hepatocytes and vice versa, the results suggest that the first-pass elimination of this amphiphilic thrombin inhibitor is due to an active carrier-mediated transport process in the basolateral plasma membrane of rat hepatocytes, and that this transport occurs via a bile acid transport system.

The effect of the calcium antagonist verapamil on gastric acid secretion in humans.

Studies on isolated gastric glands and parietal cells suggest that calcium plays a role in the stimulus-secretion coupling of acid secretion. To determine which stimulants of human gastric secretion depend on calcium-mediated receptors, the effect of the calcium antagonist verapamil on gastric secretion was studied. Gastric secretion was stimulated by intravenous infusion of 50 micrograms/kg/h bethanechol, 10 micrograms/kg/h impromidine, 0.5 micrograms/kg/h pentagastrin, 4 mg/kg/h Ca++, or by sham feeding. Each type of stimulation was tested twice in six healthy subjects, alone and together with verapamil. Verapamil was administered as an intravenous bolus of 200 micrograms/kg followed by an infusion of 150 micrograms/kg/h. Six subjects had 2 step tests in which 4 doses of pentagastrin were administered consecutively in increasing dosage. Pentagastrin was given either alone or superimposed on a verapamil bolus followed by an intravenous infusion. Verapamil left gastric secretion stimulated by bethanechol, impromidine, or sham feeding unaffected. During Ca++ infusion verapamil caused a significant drop in volume, but not acid output. Pentagastrin-stimulated acid and volume output were significantly inhibited by intravenous verapamil, the inhibition resembling an uncompetitive pattern. Pentagastrin-stimulated gastric secretion was also inhibited by the calcium antagonist diltiazem. The results confirm earlier reports that calcium is involved in the stimulus-secretion coupling of the human gastric mucosa. Calcium appears to mediate the stimulation of acid secretion by gastrin.

Two iron-regulated cation transporters from tomato complement metal uptake-deficient yeast mutants.

Although iron deficiency poses severe nutritional problems to crop plants, to date iron transporters have only been characterized from the model plant Arabidopsis thaliana. To extend our molecular knowledge of Fe transport in crop plants, we have isolated two cDNAs (LeIRT1 and LeIRT2) from a library constructed from roots of iron-deficient tomato (Lycopersicon esculentum) plants, using the Arabidopsis iron transporter cDNA, IRTI, as a probe. Their deduced polypeptides display 64% and 62% identical amino acid residues to the IRT1 protein, respectively. Transcript level analyses revealed that both genes were predominantly expressed in roots. Transcription of LeIRT2 was unaffected by the iron status of the plant, while expression of LeIRT1 was strongly enhanced by iron limitation. The growth defect of an iron uptake-deficient yeast (Saccharomyces cerevisiae) mutant was complemented by LeIRT1 and LeIRT2 when ligated to a yeast expression plasmid. Transport assays revealed that iron uptake was restored in the transformed yeast cells. This uptake was temperature-dependent and saturable, and Fe2+ rather than Fe3+ was the preferred substrate. A number of divalent metal ions inhibited Fe2+ uptake when supplied at 100-fold or 10-fold excess. Manganese, zinc and copper uptake-deficient yeast mutants were also rescued by the two tomato cDNAs, suggesting that their gene products have a broad substrate range. The gene structure was determined by polymerase chain reaction experiments and, surprisingly, both genes are arranged in tandem with a tail-to-tail orientation.

Substrate specificity of sinusoidal bile acid and organic anion uptake systems in rat and human liver.

The Na+-dependent bile salt uptake systems Ntcp (rodents) and NTCP (human), and the Na+-independent organic anion transporters oatpl (rat) and OATP (human) mediate sinusoidal uptake of a variety of amphipathic organic compounds into hepatocytes. Their properties indicate that an overall hepatic clearance of albumin-bound compounds is mediated by a limited number of multispecific transporters with partially overlapping substrate specificities.

Polyspecific substrate uptake by the hepatic organic anion transporter Oatp1 in stably transfected CHO cells.

The rat liver organic anion transporting polypeptide (Oatp1) has been extensively characterized mainly in the Xenopus laevis expression system as a polyspecific carrier transporting organic anions (bile salts), neutral compounds, and even organic cations. In this study, we extended this characterization using a mammalian expression system and confirm the basolateral hepatic expression of Oatp1 with a new antibody. Besides sulfobromophthalein [Michaelis-Menten constant (Km) of approximately 3 microM], taurocholate (Km of approximately 32 microM), and estradiol- 17beta-glucuronide (Km of approximately 4 microM), substrates previously shown to be transported by Oatp1 in transfected HeLa cells, we determined the kinetic parameters for cholate (Km of approximately 54 microM), glycocholate (Km of approximately 54 microM), estrone-3-sulfate (Km of approximately 11 microM), CRC-220 (Km of approximately 57 microM), ouabain (Km of approximately 3,000 microM), and ochratoxin A (Km of approximately 29 microM) in stably transfected Chinese hamster ovary (CHO) cells. In addition, three new substrates, taurochenodeoxycholate (Km of approximately 7 microM), tauroursodeoxycholate (Km of approximately 13 microM), and dehydroepiandrosterone sulfate (Km of approximately 5 microM), were also investigated. The results establish the polyspecific nature of Oatp1 in a mammalian expression system and definitely identify conjugated dihydroxy bile salts and steroid conjugates as high-affinity endogenous substrates of Oatp1.

Titer determination of Ad5 in blood: a cautionary note.

Recombinant adenoviruses are presently the most efficient in vivo gene transfer system available. Targeting single organs or large tumors by adenoviral vectors requires an intravascular route of application. During the first pass of viral particles through the vascular bed of the target tissue, virus uptake is not quantitative and indefinite amounts of particles leak into circulation. To determine the amount of leaking particles and to calculate organ-specific uptake (in-/outflow ratio), it is necessary to titrate virus particles directly in blood. In preclinical and clinical trials titration is currently mostly done with blood plasma instead of full blood. However, this technique provides valid results only as long as there is no affinity between adenovirus particles and erythrocytes. In this study we demonstrate that Ad5 particles, as mostly employed for gene therapy, have a strong affinity to human erythrocytes. At 60 min after coincubation of human erythrocytes and Ad5 particles, more than 98% of the particles are attached to the surface of erythrocytes. Therefore, ignoring the amount of red cell bound particles by performing titration in plasma leads to severe miscalculation of organ-specific transfer rates or virus circulation half-life. The biological impact of an increased affinity between virus particles and erythrocytes will be discussed.

The peptide-based thrombin inhibitor CRC 220 is a new substrate of the basolateral rat liver organic anion-transporting polypeptide.

The peptidomimetic thrombin inhibitor CRC 220, 4-methoxy-2,3,6-trimethylphenylsulfonyl-L-aspartyl-D-4-amidinop henylalanyl- piperidide, is taken up into isolated rat hepatocytes through active, carrier-mediated transport. This uptake is inhibited by bile acids. Functional expression in Xenopus laevis oocytes was performed to identify the transport system responsible for the hepatocellular CRC 220 uptake. Injection of poly(A)+RNA in X. laevis oocytes resulted in a two- to three-times higher uptake of CRC 220, compared with uninjected or water-injected control oocytes. Taurocholate (200 mumol/L) inhibited this uptake completely. No uptake of the peptidomimetic thrombin inhibitor was observed, when X. laevis oocytes were injected with complementary RNA (cRNA) encoding either the cloned rat liver Na(+)-dependent taurocholate transporter Ntcp, the renal oligopeptide carrier rhaPT or the intestinal oligopeptide transporter PepT1. However, after injection of cRNA of the cloned rat liver Na(+)-independent organic anion transporting polypeptide oatp, a specific and saturable CRC 220 uptake was observed (Michaelis-Menten constant 29.5 mumol/L). Cis-inhibition with known oatp-substrates, e.g., 20 mumol/L Bromsulphalein (BSP), 2007 mumol/L taurocholate and 2007 mumol/L cholate, occurred in oatp-expressing X. laevis oocytes, whereas substrates of the two peptide carriers as well as dipeptide- and single-amino acid constituents of the thrombin inhibitor itself lacked any significant inhibitory effects. These data show that the modified dipeptide CRC 220 is a highly selective substrate of the organic anion transporting polypeptide oatp in the basolateral plasma membrane of rat hepatocytes.

Substrate specificity of the rat liver Na(+)-bile salt cotransporter in Xenopus laevis oocytes and in CHO cells.

It has been proposed that the hepatocellular Na(+)-dependent bile salt uptake system exhibits a broad substrate specificity in intact hepatocytes. In contrast, recent expression studies in mammalian cell lines have suggested that the cloned rat liver Na(+)-taurocholate cotransporting polypeptide (Ntcp) may transport only taurocholate. To characterize its substrate specificity Ntcp was stably transfected into Chinese hamster ovary (CHO) cells. These cells exhibited saturable Na(+)-dependent uptake of [3H]taurocholate [Michaelis constant (K(m)) of approximately 34 microM] that was strongly inhibited by all major bile salts, estrone 3-sulfate, bumetanide, and cyclosporin A. Ntcp cRNA-injected Xenopus laevis oocytes and the transfected CHO cells exhibited saturable Na(+)-dependent uptake of [3H]taurochenodeoxycholate (Km of approximately 5 microM), [3H]tauroursodeoxycholate (Km of approximately 14 microM), and [14C]glycocholate (Km of approximately 27 microM). After induction of gene expression by sodium butyrate, Na(+)-dependent transport of [3H]estrone 3-sulfate (Km of approximately 27 microM) could also be detected in the transfected CHO cells. However, there was no detectable Na(+)-dependent uptake of [3H]bumetanide or [3H]cyclosporin A. These results show that the cloned Ntcp can mediate Na(+)-dependent uptake of all physiological bile salts as well as of the steroid conjugate estrone 3-sulfate. Hence, Ntcp is a multispecific transporter with preference for bile salts and other anionic steroidal compounds.

What we have learned about bumetanide and the concept of multispecific bile acid/drug transporters from the liver.

Bumetanide is a weak organic acid which is transported into hepatocytes by a transport system that is related neither to the cloned sodium-dependent taurocholate cotransporting polypeptide Ntcp nor to the cloned organic anion transporting polypeptide oatp. Bumetanide is known to be transported in the kidney by a multispecific organic anion transporter which is the pAH-transporter from the proximal tubule cell. In the liver, bumetanide uptake competes with bile acid uptake, indicating a functionally related multispecific transporter for bile acids and drugs in hepatocytes. This multispecific bile acid transporter MBAT has not been cloned yet. When basolateral membranes were photoaffinity labeled with [3H]bumetanide, several bumetanide binding proteins were separated and identified after protein sequencing from two-dimensional electrophoresis gels.