Olive leaf extract counteracts cell proliferation and cyst growth in an in vitro model of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive enlargement of kidney cysts, leading to chronic kidney disease. Since the available treatment for ADPKD is limited, there is emerging interest for natural compounds as potential therapeutic candidates. The aim of our study was to investigate whether an olive leaf extract may be able to counteract the cyst growth in an in vitro model of ADPKD. We treated WT9-12 cells with an olive leaf extract (OLE). In monolayer culture we evaluated cell viability by the MTT assay, protein expression by western-blot analysis and apoptosis by DNA laddering and TUNEL assays. For functional studies we used transient transfection and ChIP assays. Intracellular calcium measurement was performed with a spectrofluorimeter using a fluorescent probe. 3D-cell-culture was used for cyst growth studies. OLE reduced the WT9-12 cell growth rate and affected intracellular signaling due to high c-AMP levels, as OLE reduced PKA levels, enhanced p-AKT, restored B-Raf-inactivation and down-regulated p-ERK. We elucidated the molecular mechanism by which OLE, via Sp1, transactivates the p21WAF1/Cip1 promoter, whose levels are down-regulated by mutated PKD1. We demonstrated that p-AKT up-regulation also played a crucial role in the OLE-induced anti-apoptotic effect and that OLE ameliorated intracellular calcium levels, the primary cause of ADPKD. Finally, using a 3D-cell-culture model we observed that OLE reduced the cyst size. Therefore, multifaceted OLE may be considered a new therapeutic approach for ADPKD treatment.

Kinetic characterization of the reconstituted ornithine carrier from rat liver mitochondria.

The ornithine carrier was purified from rat liver mitochondria and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-2. The efficiency of reconstitution was optimized with respect to the concentration of protein and phospholipid, the Triton X-100/phospholipid ratio, the Amberlite/detergent ratio and the number of passages through a single Amberlite column. The activity of the carrier was influenced by the phospholipid composition of the liposomes, increasing in the presence of acidic phospholipids and decreasing in the presence of dioleoylphosphatidylcholine. In the reconstituted system the incorporated ornithine carrier catalyzed a first-order reaction of ornithine/ornithine or ornithine/citrulline exchange. The maximum transport rate of external [14C]ornithine was 3.2 mmol/min per g protein at 25 degrees C. This value was independent of the type of substrate present at the external or internal space of the liposomes (ornithine, citrulline and lysine). The half-saturation constant (Km) was 0.16 mM for ornithine, 1.2 mM for lysine and 3.6 mM for citrulline. The activation energy of the ornithine/ornithine exchange reaction was 89 kJ/mol. The rate of exchange had a pH optimum at 8 and was inhibited by cations.

Purification and reconstitution of two anion carriers from rat liver mitochondria: the dicarboxylate and the 2-oxoglutarate carrier.

Two anion-transporting systems, i.e., the dicarboxylate carrier and the 2-oxoglutarate carrier, have been purified from rat liver mitochondria and functionally identified. The dicarboxylate carrier has been isolated in active form by hydroxyapatite chromatography after partial removal of the solubilizing detergent Triton X-114 from the mitochondrial extract. The SDS gel electrophoresis of this preparation consists mainly of one protein band with an apparent Mr of 28,000, identified as the dicarboxylate carrier. Complete purification of the 28 kDa protein in inactive form has been achieved by sequential chromatography on hydroxyapatite and Celite followed by SDS extraction of the retained protein. The 2-oxoglutarate carrier has been purified by hydroxyapatite chromatography after extensive removal of Triton X-114 from the detergent extract. SDS gel electrophoresis of the purified fraction shows a single band with an apparent Mr of 32,500. When reconstituted into liposomes, the functional properties of the two isolated carrier proteins resemble closely those of the dicarboxylate and the 2-oxoglutarate transport systems characterized in mitochondria.

The reconstituted carnitine carrier from rat liver mitochondria: evidence for a transport mechanism different from that of the other mitochondrial translocators.

The transport mechanism of the reconstituted carnitine carrier purified from rat liver mitochondria was investigated kinetically. The half-saturation constant (Km) for carnitine on the internal side of the liposomal membrane (8.7 mM) was found to be much higher than that determined for the external surface (0.45 mM). The exclusive presence of a single transport affinity for carnitine on each side of the membrane indicated a unidirectional insertion of the carnitine carrier into the proteoliposomes, most probably right-side-out with respect to mitochondria. Under these defined conditions bisubstrate initial velocity studies of homologous (carnitine/carnitine) and heterologous (carnitine/acylcarnitine) antiport were performed by varying both the internal and external substrate concentrations. The kinetic patterns obtained showed that the ratio Km/Vmax is not influenced by the second (non-varied) substrate, which indicates a ping-pong mechanism. The carnitine carrier thus differs from all other mitochondrial carriers analyzed so far in the reconstituted state, for which a common sequential type of reaction mechanism has been found.

Purification of reconstitutively active alpha-oxoglutarate carrier from pig heart mitochondria.

The alpha-oxoglutarate carrier from pig heart mitochondria has been solubilized with Triton X-114 and purified by chromatography on hydroxyapatite and celite in the presence of cardiolipin. When applied to SDS gel electrophoresis, the purified protein consists of only a single protein band with an apparent Mr of 31.5 kDa. It corresponds to band 4 of the five protein bands previously identified in the hydroxyapatite pass-through of Triton X-114 solubilized heart mitochondria (Bisaccia, F. and Palmieri, F. (1984) Biochim. Biophys. Acta 766, 386-394). When reconstituted into liposomes the alpha-oxoglutarate transport protein catalyzes a phthalonate-sensitive alpha-oxoglutarate/alpha-oxoglutarate exchange. It is purified 250-fold with a recovery of 62% and a protein yield of 0.1% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., the requirements for a counteranion, the substrate specificity and the inhibitor sensitivity, are similar to those described for alpha-oxoglutarate transport in mitochondria.

Identification and purification of the carnitine carrier from rat liver mitochondria.

The carnitine carrier from rat liver mitochondria, solubilized in Triton X-100 and partially purified on hydroxyapatite, was identified and completely purified by specific elution from celite in the presence of cardiolipin. On SDS-gel electrophoresis, the purified celite fraction consisted of a single band with an apparent Mr of 32,500. When reconstituted into liposomes the carnitine transport protein catalyzed an N-ethylmaleimide-sensitive carnitine/carnitine exchange. It was purified 970-fold with a recovery of 43% and a protein yield of 0.04% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., requirement for a countersubstrate, substrate specificity and inhibitor sensitivity, were similar to those of the carnitine transport system as characterized in intact mitochondria.

Characterization of the unidirectional transport of carnitine catalyzed by the reconstituted carnitine carrier from rat liver mitochondria.

The carnitine carrier from rat liver mitochondria was purified by chromatography on hydroxyapatite and celite and reconstituted in egg yolk phospholipid vesicles by adsorbing the detergent on polystyrene beads. In the reconstituted system, in addition to the carnitine/carnitine exchange, the purified protein catalyzed a uni-directional transport (uniport) of carnitine measured as uptake into unloaded proteoliposomes as well as efflux from prelabelled proteoliposomes. In both cases the reaction followed a first-order kinetics with a rate constant of 0.023-0.026 min-1. Besides carnitine, also acylcarnitines were transported in the uniport mode. N-Ethylmaleimide inhibited the uni-directional transport of carnitine completely. The uniport of carnitine is not influenced by the delta pH and the electric gradient across the membrane. The activation energy for uniport was 115 kJ/mol and the half-saturation constant on the external side of the proteoliposomes was 0.53 mM. The maximal rate of the uniport at 25 degrees C was 0.2 mumol/min per mg protein, i.e. about 10 times lower than that of the reconstituted carnitine transport in exchange mode.

Kinetics of the reconstituted dicarboxylate carrier from rat liver mitochondria.

The dicarboxylate carrier from rat liver mitochondria was purified by the Amberlite/hydroxyapatite procedure and reconstituted in egg yolk phospholipid vesicles by removing the detergent with Amberlite. The efficiency of reconstitution was optimized with respect to the ratio of detergent/phospholipid, the concentration of phospholipid and the number of Amberlite column passages. In the reconstituted system the incorporated dicarboxylate carrier catalyzed a first-order reaction of malate/phosphate exchange. V of the reconstituted malate/phosphate exchange was determined to be 6000 mumol/min per g protein at 25 degrees C. This value was independent of the type of substrate present at the external or internal space of the liposomes (malate, phosphate or malonate). The half-saturation constant was 0.49 mM for malate, 0.54 mM for malonate and 1.41 mM for phosphate. The activation energy of the exchange reaction was determined to be 95.8 kJ/mol. The transport was independent of the external pH in the range between pH 6 and 8.

Kinetic discrimination of two substrate binding sites of the reconstituted dicarboxylate carrier from rat liver mitochondria.

The kinetic interaction of various substrates and inhibitors with the dicarboxylate carrier from rat liver mitochondria was investigated using the isolated and reconstituted carrier protein. Due to their inhibitory interrelation the ligands could be divided into two classes: dicarboxylates, sulphate, sulphite and butylmalonate on the one hand and phosphate, thiosulphate and arsenate on the other. The mutual inhibition of substrates or inhibitors taken from one single class was found to be competitive, whereas the kinetic interaction of ligands when taken from the two different classes could be described as purely non-competitive. The half-saturation transport constants Km and the corresponding inhibition constants Ki of one single ligand, either used as substrate or as inhibitor, respectively, were found to be very similar. These kinetic data strongly support the presence of two different binding sites at the dicarboxylate carrier for the two different classes of substrates considering the external side of the reconstituted protein. When these two sites were saturated simultaneously with malate and phosphate, the turnover of the carrier was considerably reduced, hence indicating that a non-catalytic ternary complex is formed by the two substrates and the carrier molecule.

Kinetic characterization of the reconstituted carnitine carrier from rat liver mitochondria.

The carnitine carrier was purified from rat liver mitochondria and reconstituted into liposomes by removing the detergent from mixed micelles by Amberlite. Optimal transport activity was obtained with 1 microgram/ml and 12.5 mg/ml of protein and phospholipid concentration, respectively, with a Triton X-100/phospholipid ratio of 1.8 and with 16 passages through the same Amberlite column. The activity of the carrier was influenced by the phospholipid composition of the liposomes, being increased in the presence of cardiolipin and decreased in the presence of phosphatidylinositol. In the reconstituted system the incorporated carnitine carrier catalyzed a carnitine/carnitine exchange which followed a first-order reaction. The maximum transport rate of external [3H]carnitine was 1.7 mmol/min per g protein at 25 degrees C and was independent of the type of countersubstrate. The half-saturation constant (Km) for carnitine was 0.51 mM. The affinity of the carrier for acylcarnitines was in the microM range and depended on the carbon chain length. The activation energy of the carnitine/carnitine exchange was 133 kJ/mol. The carrier function was independent of the pH in the range between 6 and 8 and was inhibited at pH below 6.

Kinetics of the reconstituted 2-oxoglutarate carrier from bovine heart mitochondria.

The 2-oxoglutarate carrier from the inner membrane of bovine heart mitochondria was purified by chromatography on hydroxyapatite/celite and reconstituted with egg yolk phospholipid vesicles by the freeze-thaw-sonication technique. In the reconstituted system the incorporated 2-oxoglutarate carrier catalyzed a first-order reaction of 2-oxoglutarate/2-oxoglutarate exchange. The substrate affinity for 2-oxoglutarate was determined to be 65 +/- 18 microM (15 determinations) and the maximum exchange rate at 25 degrees C reaches 4000-22,000 mumol/min per g protein, in dependence of the particular reconstitution conditions. The activation energy of the exchange reaction is 54.3 kJ/mol. The transport is independent of pH in the range between 6 and 8. When the first fraction of the hydroxyapatite/celite column eluate was used for reconstitution, besides the 2-oxoglutarate/2-oxoglutarate exchange, a significant activity of unidirectional uptake was observed. This activity may be due to a population of the carrier protein which is in a different state.

Kinetic mechanism of antiports catalyzed by reconstituted ornithine/citrulline carrier from rat liver mitochondria.

The transport mechanism of the reconstituted ornithine/citrulline carrier purified from rat liver mitochondria was investigated kinetically. A complete set of half-saturation constants (K(m)) was established for ornithine, citrulline and H(+) on both the external and internal side of the liposomal membrane. The internal affinity for ornithine was much lower than that determined on the external surface. The exclusive presence of a single transport affinity for ornithine on each side of the membrane indicated a unidirectional insertion of the ornithine/citrulline carrier into liposomes, probably right-side-out with respect to mitochondria. Two-reactant initial velocity studies of the homologous (ornithine/ornithine) and heterologous (ornithine/citrulline) exchange reactions resulted in a kinetic pattern which is characteristic of a simultaneous antiport mechanism. This type of mechanism implies that the carrier forms a ternary complex with the substrates before the transport reaction occurs. A quantitative analysis of substrate interaction revealed that rapid-equilibrium random conditions were fulfilled, characterized by a fast and independent binding of internal and external substrates.

Kinetic characterization of the reconstituted dicarboxylate carrier from mitochondria: a four-binding-site sequential transport system.

The mitochondrial antiport carriers form a protein family with respect to their structure and function. The kinetic antiport mechanism, being of the sequential type, shows that the dicarboxylate carrier also belongs to this family. This was demonstrated by bireactant initial velocity studies of the purified and reconstituted carrier protein. The transport affinity of the carrier for the internal substrate was largely independent of the external substrate concentration and vice versa, whereas the carrier's apparent Vmax rose with increasing saturation of internal and external binding sites. Thus, the carrier forms a catalytic ternary complex with one internal and one external substrate molecule. As compared to other mitochondrial antiport carriers, however, the situation with the dicarboxylate carrier is more complex. On each membrane side of the protein two separate binding sites exist, one specific for phosphate (or its analogue phenyl phosphate), the other specific for dicarboxylate (or butyl malonate), that can be occupied by the respective substrates without mutual interference. This became evident from the non-competitive interaction of these substrates (or analogues) with the carrier. The two external, but not the two internal binding sites could be saturated simultaneously with phosphate and malate, thereby causing inhibition of transport. All four binding sites must be associated with the same translocation pathway through the carrier protein, since the sequential antiport mechanism held true for the phosphate/malate heteroexchange as well as for the malate/malate or phosphate/phosphate homoexchange.

The mitochondrial carnitine carrier: characterization of SH-groups relevant for its transport function.

The transport function of the purified and reconstituted carnitine carrier from rat liver mitochondria was correlated to modification of its SH-groups by various reagents. The exchange activity and the unidirectional transport, both catalyzed by the carnitine carrier, were effectively inhibited by N-ethylmaleimide and submicromolar concentrations of mercurial reagents, e.g., mersalyl and p-(chloromercuri)benzenesulfonate. When 1 microM HgCl2 or higher concentrations of the above mentioned mercurials were added, another transport mode of the carrier was induced. After this treatment, the reconstituted carnitine carrier catalyzed unidirectional substrate-efflux and -influx with significantly reduced substrate specificity. Control experiments in liposomes without carrier or with inactivated carrier protein proved the dependence of this transport activity on the presence of active carnitine carrier. The mercurial-induced uniport correlated with inhibition of the 'physiological' functions of the carrier, i.e., exchange and substrate specific unidirectional transport. The effect of consecutive additions of various reagents including N-ethylmaleimide, mercurials, Cu(2+)-phenanthroline and diamide on the transport function revealed the presence of at least two different classes of SH-groups. N-Ethylmaleimide blocked the carrier activity by binding to SH-groups of one of these classes. At least one of these SH-groups could be oxidized by the reagents forming S-S bridges. Besides binding to the class of SH-groups to which N-ethylmaleimide binds, mercurials also reacted with SH-groups of the other class. Modification of the latter led to the induction of the efflux-type of carrier activity characterized by loss of substrate specificity.

Purification of the mitochondrial carnitine carrier by chromatography on hydroxyapatite and celite.

The carnitine carrier from rat liver mitochondria has been extracted with Triton X-100 ad partially purified by chromatography on hydroxyapatite and celite. During purification the activity of the carrier was monitored by functional reconstitution into liposomes. The purified fraction is 250-fold enriched with respect to the N-ethylmaleimide-sensitive carnitine/carnitine transport activity. The substrate specificity and the inhibitor sensitivity of carnitine transport in liposomes resemble closely those described for the transport of carnitine in mitochondria.