Cell-permeable succinate prodrugs rescue mitochondrial respiration in cellular models of acute acetaminophen overdose.

Acetaminophen is one of the most common over-the-counter pain medications used worldwide and is considered safe at therapeutic dose. However, intentional and unintentional overdose accounts for up to 70% of acute liver failure cases in the western world. Extensive research has demonstrated that the induction of oxidative stress and mitochondrial dysfunction are central to the development of acetaminophen-induced liver injury. Despite the insight gained on the mechanism of acetaminophen toxicity, there still is only one clinically approved pharmacological treatment option, N-acetylcysteine. N-acetylcysteine increases the cell's antioxidant defense and protects liver cells from further acetaminophen-induced oxidative damage. Because it primarily protects healthy liver cells rather than rescuing the already injured cells alternative treatment strategies that target the latter cell population are warranted. In this study, we investigated mitochondria as therapeutic target for the development of novel treatment strategies for acetaminophen-induced liver injury. Characterization of the mitochondrial toxicity due to acute acetaminophen overdose in vitro in human cells using detailed respirometric analysis revealed that complex I-linked (NADH-dependent) but not complex II-linked (succinate-dependent) mitochondrial respiration is inhibited by acetaminophen. Treatment with a novel cell-permeable succinate prodrug rescues acetaminophen-induced impaired mitochondrial respiration. This suggests cell-permeable succinate prodrugs as a potential alternative treatment strategy to counteract acetaminophen-induced liver injury.

Quantification of a cocrystal and its dissociated compounds in solid dosage form using transmission Raman spectroscopy.

The performance of transmission Raman spectroscopy (TRS) for quantifying a cocrystal and its dissociation in solid dosage form was investigated. Some tablets containing 0%-20% (w/w) of a cocrystal of carbamazepine (CBZ)/succinic acid (SUC), 0%-4% of CBZ, 0%-4% of SUC, and 75%-99% of D-mannitol were prepared. The Raman spectra of these tablets were preprocessed using the standard normal variate (SNV) or multiplicative scatter correction (MSC) as well as the Savitzky Golay second derivative, and then, these were used to generate calibration models using partial least squares (PLS) regression. The performance of the model was superior when the MSC preprocessing spectra of the cocrystal between 200 and 1800 cm-1 were used for calibration. The determination coefficient of the PLS calibration curve for the CBZ/SUC cocrystal between 200 and 1800 cm-1 with MSC was 0.97, root mean square error of cross validation (RMSECV) was 1.16, and root mean square error of prediction (RMSEP) was 1.10. As in the case of the CBZ/SUC cocrystal, the performance of the model was superior when the MSC preprocessing spectra of CBZ and SUC between 200 and 1800 cm-1 were used for calibration. These data suggest that TRS is useful for quantifying a cocrystal and its dissociation compounds in solid dosage forms.

Cation instructed steroidal prodrug supramolecular hydrogel.

In the present study, we propose an ionic coordination strategy for the design of a steroidal prodrug supramolecular hydrogel. The hydrogel composed of nanofibril networks formed spontaneously by the introduction of divalent cations (e.g., Mg2+, Ca2+, Zn2+ and Fe2+) and NH4+ to a succinated dexamethasone (Dex-SA) aqueous solution at room temperature. The formation of the nanofibril structure was dominantly driven by the ionic coordination with the assistance of a delicate balance of multiple noncovalent interactions. A rheological analysis indicated that the formed Ca2+/Dex-SA supramolecular hydrogel exhibits dominant elastic and thixotropic properties. The formed Ca2+/Dex-SA supramolecular hydrogel allowed the gradual release of Dex and Dex-SA in vitro, and the drug release behaviour can be finely tuned by changing the Ca2+ concentration. Storage stability studies showed that Dex-SA in hydrogel underwent an apparent chemical decomposition at 4 °C and 37 °C. In contrast, the Dex-SA xerogel was quite stable without any obvious chemical decomposition of Dex-SA in storage at -20 °C for 35 days, and it was able to turn into a hydrogel again within one minute after rehydration. The formed Ca2+/Dex-SA supramolecular hydrogel caused negligible cytotoxicity against HCEC and L-929 cells at drug concentrations up to 2 mM, as indicated by the in vitro cytotoxicity tests. Additionally, the proposed Ca2+/Dex-SA supramolecular hydrogel displayed a comparable anti-inflammatory efficacy with Dexp via the downregulation of NO, TNF-α and IL-6 expression in lipopolysaccharide (LPS)-activated RAW264.7 macrophage. Overall, the cation instructed steroidal prodrug supramolecular hydrogel might be a promising ophthalmic drug delivery system for anti-inflammatory therapy.

Synthesis, stability and bioavailability of astaxanthin succinate diester.

BACKGROUND: We synthesized astaxanthin succinate diester (ASD), a novel astaxanthin (AST) derivate, with succinic anhydride and free AST. ASD was purified and characterized using silica gel column chromatography and spectrometry, respectively. RESULTS: The ASD final synthesis rate was 82.63%. A stability test revealed a high AST and ASD retention rate at pH 5.0-7.0. ASD showed better stability than did AST under acidic conditions. Both sample ions showed lower retention rates under Fe2+ and Fe3+ states. The ASD metabolic curve showed serum and liver area under the curve from 0 h to time t (AUC0-t ) values of 45.05 ± 4.58 and 120.38 ± 23.66 µg h-1  mL-1 , respectively. The long-term accumulation was significantly higher in the ASD group than in the AST group, which showed higher accumulation in the heart, muscle and spleen than in other tissues in vivo. CONCLUSION: The thermal stability and bioavailability of ASD were higher than that of the non-esterified free AST and common free AST, respectively. Additionally, AST accumulation in different tissues of the ASD group was multifold higher than that of free AST. These results prove that ASD may serve as a better source of AST for human nutrition than does free AST. © 2017 Society of Chemical Industry.

Dual 7-ethyl-10-hydroxycamptothecin conjugated phospholipid prodrug assembled liposomes with in vitro anticancer effects.

7-Ethyl-10-hydroxycamptothecin (SN38), as a highly active topoisomerase I inhibitor, is 200-2000-fold more cytotoxic than irinotecan (CPT-11) commercially available as Camptosar®. However, poor solubility and low stability extensively restricted its clinical utility. In this report, dual SN38 phospholipid conjugate (Di-SN38-PC) prodrug based liposomes were developed in order to compact these drawbacks. Di-SN38-PC prodrug was first synthesized by inhomogeneous conjugation of two SN38-20-O-succinic acid molecules with L-α-glycerophosphorylcholine (GPC). The assembly of the prodrug was carried out without any excipient by using thin film method. Dynamic light scattering (DLS), transmission electron microscope (TEM) and cryogenic transmission electron microscopy (cyro-TEM) characterization indicated that Di-SN38-PC can form spherical liposomes with narrow particle size (<200nm) and negatively charged surface (-21.6±3.5mV). The loading efficiency of SN38 is 65.2 wt.% after a simple calculation. In vitro release test was further performed in detail. The results demonstrated that Di-SN38-PC liposomes were stable in neutral environment but degraded in a weakly acidic condition thereby released parent drug SN38 effectively. Cellular uptake studies reflected that the liposomes could be internalized into cells more significantly than SN38. In vitro antitumor activities were finally evaluated by MTT assay, colony formation assay, flow cytometry, RT-PCR analysis and Western Blot. The results showed that Di-SN38-PC liposomes had a comparable cytotoxicity with SN38 against MCF-7 and HBL-100, and a selective promotion of apoptosis of tumor cells. Furthermore, a pharmacokinetics test showed that Di-SN38-PC liposomes had a longer circulating time in blood compared with the parent drug. All the results indicate that Di-SN38-PC liposomes are an effective delivery system of SN38.

RELATIVE BIOAVAILABILITY STUDY OF SUCCINIC ACID COCRYSTAL TABLET AND MARKETED CONVENTIONAL IMMEDIATE RELEASE TABLET FORMULATION OF CARBAMAZEPINE 200 MG IN RABBITS.

A single-dose study was performed to observe the bioequivalence of the newly formulated carbamazepine-succinic cocrystal (CBZ-SUC) immediate release tablet (F(1)) with marketed immediate release formulation Epitoll 200 mg tablet (F(0)). In this study on albino rabbits, the plasma levels resulting from 250 mg cocrystal equivalent to 200 mg of carbamazepine and conventional tablets 200 mg immediate release tablets were compared. An open-label, randomized 2 x 2 crossover study design, with a 1-week washout period, was used. Carbamazapine (CBZ) plasma concentrations were determined by a high-performance liquid chromatography validated method using ultraviolet detection. CBZ plasma levels were measured at predose and various postdose time points up to 72 h and the following pharmacokinetic parameters were used for evaluation: area under the curve (AUC), maximum plasma drug concentration (C(max)), time to achieve C(max), and elimination rate constant. (K(e)). By applying paired t-test to AUC2(0-72) (calculated by linear trapezoidal rule), the experimental formulation F(1), was found to have statistically significant (***p < 0.05) improvement in bioavailability of CBZ. However, these statistical differences do not have practical implications and the two formulations (F(0) and F(1)) were found to be bioequivalent as the relative bioavailability of both formulations (106.9%) falls with- in the acceptable FDA set range of two bioequivalent products 80-125%.

Salt and cocrystals of sildenafil with dicarboxylic acids: solubility and pharmacokinetic advantage of the glutarate salt.

Sildenafil is a drug used to treat erectile dysfunction and pulmonary arterial hypertension. Because of poor aqueous solubility of the drug, the citrate salt, with improved solubility and pharmacokinetics, has been marketed. However, the citrate salt requires an hour to reach its peak plasma concentration. Thus, to improve solubility and bioavailability characteristics, cocrystals and salts of the drug have been prepared by treating aliphatic dicarboxylic acids with sildenafil; the N-methylated piperazine of the drug molecule interacts with the carboxyl group of the acid to form a heterosynthon. Salts are formed with oxalic and fumaric acid; salt monoanions are formed with succinic and glutaric acid. Sildenafil forms cocrystals with longer chain dicarboxylic acids such as adipic, pimelic, suberic, and sebacic acids. Auxiliary stabilization via C-H···O interactions is also present in these cocrystals and salts. Solubility experiments of sildenafil cocrystal/salts were carried out in 0.1N HCl aqueous medium and compared with the solubility of the citrate salt. The glutarate salt and pimelic acid cocrystal dissolve faster than the citrate salt in a two hour dissolution experiment. The glutarate salt exhibits improved solubility (3.2-fold) compared to the citrate salt in water. Solubilities of the binary salts follow an inverse correlation with their melting points, while the solubilities of the cocrystals follow solubilities of the coformer. Pharmacokinetic studies on rats showed that the glutarate salt exhibits doubled plasma AUC values in a single dose within an hour compared to the citrate salt. The high solubility of glutaric acid, in part originating from the strained conformation of the molecule and its high permeability, may be the reason for higher plasma levels of the drug.

Preclinical pharmacokinetic analysis of armillarisin succinate ester in mouse plasma and tissues by LC-MS/MS.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated to determine the concentration of armillarisin succinate ester in mouse plasma and tissues, used for preclinical evaluation. Bavachin was employed as the internal standard. Separation was performed on a 3.5 µm Zorbax SB-C(18) column (30 × 2.1 mm), with a mobile phase consisting of methanol and aqueous 20 mm ammonium acetate. Both analyte and internal standard were determined using electrospray ionization and the MS data acquisition was via selected ion monitoring in negative scanning mode. Quantification was performed using the transitions m/z 333 → 233 and 323 → 221 for armillarisin succinate ester and internal standard, respectively. The method was validated with respect to linearity, accuracy, precision, recovery and stability. This assay has been successfully applied to a pharmacokinetic and tissue distribution study after intravenous injection of ASE in mouse in a dose of 10 mg/kg.

[Intestinal absorption of enteric coating potassium sodium dehydroandroan drographolide succinate pellets in rat and in vivo pharmacokinetics study].

OBJECTIVE: To develop a method for determination the content of Potassium Sodium Dehydroandroan drographolide Succinate (PSDS) in rat intestinal contents and plasma and investigate the intestinal absorption of PSDS pellets in rat and in vivo pharmacokinetics of PSDS pellets. METHODS: The content of PSDS in rat intestinal contents and plasma was determined by HPLC. In vivo pharmacokinetic properties and intestinal absorption of PSDS pellets in rat were investigated. RESULTS: Two hours after administration, pellets were not found in the small intestine and large intestine, four hours after administration, the largest number of pellets were found in the small intestine and the concentration of PSDS was the highest in the intestinal contents (3593.13 microg). The characteristics of plasma concentration-time curve was consistent with a single compartment model. The main drug pharmacokinetic parameters were calculated. t1/2, T(max), C(max) and AUC were 2.69 h, 5 h, 3.02 microg/mL and 6.42 microg x h/mL, respectively. CONCLUSION: PSDS has a good absorption in the rat small intestine and it is feasible to prepare PSDS enteric-coated pellets for oral administration.

Methylmalonate inhibits succinate-supported oxygen consumption by interfering with mitochondrial succinate uptake.

The effect of methylmalonate (MMA) on mitochondrial succinate oxidation has received great attention since it could present an important role in energy metabolism impairment in methylmalonic acidaemia. In the present work, we show that while millimolar concentrations of MMA inhibit succinate-supported oxygen consumption by isolated rat brain or muscle mitochondria, there is no effect when either a pool of NADH-linked substrates or N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD)/ascorbate were used as electron donors. Interestingly, the inhibitory effect of MMA, but not of malonate, on succinate-supported brain mitochondrial oxygen consumption was minimized when nonselective permeabilization of mitochondrial membranes was induced by alamethicin. In addition, only a slight inhibitory effect of MMA was observed on succinate-supported oxygen consumption by inside-out submitochondrial particles. In agreement with these observations, brain mitochondrial swelling experiments indicate that MMA is an important inhibitor of succinate transport by the dicarboxylate carrier. Under our experimental conditions, there was no evidence of malonate production in MMA-treated mitochondria. We conclude that MMA inhibits succinate-supported mitochondrial oxygen consumption by interfering with the uptake of this substrate. Although succinate generated outside the mitochondria is probably not a sig-nificant contributor to mitochondrial energy generation, the physiopathological implications of MMA-induced inhibition of substrate transport by the mitochondrial dicarboxylate carrier are discussed.

Towards hyperpolarized (13)C-succinate imaging of brain cancer.

We describe a novel (13)C enriched precursor molecule, sodium 1-(13)C acetylenedicarboxylate, which after hydrogenation by PASADENA (Parahydrogen and Synthesis Allows Dramatically Enhanced Nuclear Alignment) under controlled experimental conditions, becomes hyperpolarized (13)C sodium succinate. Fast in vivo 3D FIESTA MR imaging demonstrated that, following carotid arterial injection, the hyperpolarized (13)C-succinate appeared in the head and cerebral circulation of normal and tumor-bearing rats. At this time, no in vivo hyperpolarized signal has been localized to normal brain or brain tumor. On the other hand, ex vivo samples of brain harvested from rats bearing a 9L brain tumor, 1 h or more following in vivo carotid injection of hyperpolarized (13)C sodium succinate, contained significant concentrations of the injected substrate, (13)C sodium succinate, together with (13)C maleate and succinate metabolites 1-(13)C-glutamate, 5-(13)C-glutamate, 1-(13)C-glutamine and 5-(13)C-glutamine. The (13)C substrates and products were below the limits of NMR detection in ex vivo samples of normal brain consistent with an intact blood-brain barrier. These ex vivo results indicate that hyperpolarized (13)C sodium succinate may become a useful tool for rapid in vivo identification of brain tumors, providing novel biomarkers in (13)C MR spectral-spatial images.

Functional roles of cationic amino acid residues in the sodium-dicarboxylate cotransporter 3 (NaDC-3) from winter flounder.

In the present study, we determined the functional role of 15 positively charged amino acid residues at or within 1 of the predicted 11 transmembrane helixes of the flounder renal sodium-dicarboxylate cotransporter fNaDC-3. Using site-directed mutagenesis, histidine (H), lysine (K), and arginine (R) residues of fNaDC-3 were replaced by alanine (A), isoleucine (I), or leucine (L). Most mutants showed sodium-dependent, lithium-inhibitable [14C]succinate uptake and, in two-electrode voltage-clamp (TEVC) experiments, Km and DeltaI(max) values comparable to wild-type (WT) fNaDC-3. The replacement of R109 and R110 by alanine and isoleucine (RR109/110AI) prevented the expression of fNaDC-3 at the plasma membrane. When the lysines at positions 232 and 235 were replaced by isoleucine (KK232/235II), the transporter was expressed but showed small transport rates and succinate-induced currents. K114I, located within transmembrane helix 4, showed [14C]succinate uptake similar to WT but relatively small inward currents. When K114 was replaced by arginine, glutamic acid (E), or glutamine (Q), all mutants were expressed at the cell surface. In [14C]succinate uptake and TEVC experiments performed simultaneously on the same oocytes, uptake was similar to or higher than WT, whereas succinate-induced currents were either comparable (K114R) to, or considerably smaller (K114E, K114I, K114Q) than, those evoked by WT. These results suggest that a positively charged residue at position 114 is required for electrogenic sodium-dicarboxylate cotransport.

Metabolic GHB precursor succinate binds to gamma-hydroxybutyrate receptors: characterization of human basal ganglia areas nucleus accumbens and globus pallidus.

Binding of the metabolic gamma-hydroxybutyrate (GHB) precursor succinate to NCS-382-sensitive [3H]GHB-labeled sites in crude synaptosomal or purified synaptic membrane fractions prepared from the human nucleus accumbens (NA), globus pallidus (GP) and rat forebrain has been shown. This site can be characterized by binding of ethyl hemisuccinate and gap-junction blockers, including carbenoxolone hemisuccinate and beta-GRA. There was no significant binding interaction between GABAB receptor ligands (CGP 55845, (R)-baclofen) and these [3H]GHB-labeled sites. GHB, NCS-382 and succinate binding profile of [3H]GHB-labeled sites in rat forebrain, human NA or GP synaptic membranes were similar. The synaptic fraction isolated from the rat forebrain was characterized by GHB binding inhibition constants: Ki,NCS-382 = 1.2 +/- 0.2 microM, Ki,GHB = 1.6 +/- 0.3 microM and Ki,SUCCINATE = 212 +/- 66 microM. In crude membranes containing mainly extrasynaptic membranes, distinct GHB and GABAB receptor sites were found in the NA. By contrast, extrasynaptic GABAB receptor sites of rat forebrain and GP were GHB- and succinate-sensitive, respectively. The heterogeneity of GABAB sites found in native membranes indicates GABAB receptor-dependent differences in GHB action. Based on these findings, we suggest that succinate (and possibly drugs available as succinate salt derivatives) can mimic some of the actions of GHB.

Regulation of sodium-dicarboxylate cotransporter-3 from winter flounder kidney by protein kinase C.

The sodium dicarboxylate cotransporter located at the basolateral side supplies renal proximal tubule cells with Krebs cycle intermediates and maintains the driving force for the exchange of organic anions like PAH against alpha-ketoglutarate through the organic anion transporter-1. Recently, we cloned sodium dicarboxylate cotransporter-3 from winter flounder kidney (fNaDC-3). To understand the regulation of fNaDC-3, we preincubated fNaDC-3-expressing oocytes with PMA, a PKC activator. PMA dose and time dependently inhibited fNaDC-3-mediated succinate uptake. Simultaneous preincubation of fNaDC-3-expressing oocytes with 50 nM PMA and either staurosporine or RO 31-8220 for 30 min attenuated PKC-mediated inhibition of succinate uptake. Site-directed mutagenesis of the five putative PKC sites (S7, T167, S174, T188, and S396) resulted in no change in PKC-mediated inhibition of the transporter. In electrophysiological studies performed at -60 mV, the K0.5 for succinate was not significantly affected (56 +/- 13 vs. 42 +/- 19 microM), but DeltaImax was reduced from -139 +/- 49 to -20 +/- 8 nA by PMA (50 nM, 30 min). Immunofluorescence analysis of fNaDC-3-expressing oocytes revealed that PMA leads to an endocytosis of fNaDC-3 protein. In conclusion, fNaDC-3 expressed in oocytes is downregulated by PMA through endocytosis. PKC consensus sites appear not to be important for this process.

Metabolite comparisons and the identity of nutrients translocated from symbiotic algae to an animal host.

Dinoflagellate algae of the genus Symbiodinium in symbiosis with marine animals release much of their photosynthetic carbon to the animal host. The compounds translocated to the host ('mobile compounds') were investigated by metabolite comparison as follows: a substrate was identified as a candidate mobile compound when comparable profiles of metabolites were generated from host metabolism of this substrate (supplied exogenously) and the endogenous mobile compounds. When the sea anemone Anemonia viridis was incubated with NaH14CO2 under photosynthesizing conditions, most of the radioactivity in the animal tissue was recovered from the low-molecular-mass fraction and distributed in the ratio 1:2:1 between the neutral, acidic and basic sub-fractions. Prominent 14C-labelled compounds included glucose, malate and glucose-6-phosphate. When the symbiosis was incubated with 14C-labelled glucose plus succinate or fumarate (but none of eight other substrate combinations tested), the 14C-labelled metabolites closely matched those obtained with NaH14CO2. These data suggest that glucose and succinate/fumarate (or metabolically allied compounds) may be important photosynthetic compounds transferred from the Symbiodinium cells to the tissues of A. viridis. Metabolite comparisons can be applied to study nutritional interactions in symbioses involving photosynthetic algae and, with appropriate modification, other associations between microorganisms and plants or animals.

Synthesis of an enzyme-dependent prodrug and evaluation of its potential for colon targeting.

AIM: To synthesize dexamethasone-succinate-dextran (DSD) conjugate and to evaluate the potentiality of DSD for the treatment of inflammatory bowel diseases. METHODS: Dexamethasone was attached to dextran (average molecular weight=70,400 Dalton) using succinate anhydride in an anhydrous environment catalyzed by 4-dimethylaminopyridine and 1, 1'-carbonyldiimidazole. The chemical structure of DSD was identified by UV, IR and NMR, and the in vivo drug release behavior of this prodrug was investigated after oral administration of DSD suspension. RESULTS: The DSD conjugate was obtained in two steps and the content of dexamethasone in DSD was 11.28 %. The dextran prodrug was stable in rat stomach and small intestine and negligibly absorbed from these tracts. Four to nine hours after the oral administration, most of the prodrug (>95 %) had moved to the cecum and colon, and was easily hydrolyzed by an endodextranase. Recover of dexamethasone from colon and cecum after administration of DSD conjugate was 6-12 folds higher than the recovery after administration of unmodified dexamethasone (t=2.74, P<0.05). The preferential release of free dexamethasone in cecum and colon over that in the small intestine was statistically significant (t=2.27, P<0.05). CONCLUSION: The results of this study indicate that dextran conjugates may be useful in selectively delivering glucocorticoids to the colon.

Highly water-soluble matrix metalloproteinases inhibitors and their effects in a rat adjuvant-induced arthritis model.

A new series of succinate-based dual inhibitors against matrix metalloproteinases (MMPs) and tumor necrosis factor alpha converting enzyme (TACE) possessing highly-water solubility was designed, synthesized, and evaluated for enzyme inhibition. Incorporating of acidic or basic functional groups at the P(2)' position afforded sufficient water solubility without significant loss of inhibitory potencies. Compound 18e, which had a guanidino group at the P(2)' position as the basic functional group, exhibited broad inhibition against target enzymes for a relatively long period in rat plasma (beta t(1/2); 2.0h) after sc administration when compared with compounds possessing acidic functional groups (18a and 18b). Consequently, the representative compound 18e together with compound 18b, Marimastat and Trocade were evaluated in the rat adjuvant-induced arthritis model, a model of chronic cartilage destruction. It is concluded that the newly synthesized highly water-soluble compound 18e showed significant activity in suppressing hindpaw swelling and the bone destruction with a minimal administration period (days 3-7).

Mitochondrial respiration and triiodothyronine concentration in liver from postpubertal and adult rats.

The purpose of this study was to investigate the decline in rat liver mitochondria respiration found in adult rats compared to younger ones, and to find a link between this respiratory impairment and a tissue hypothyroidism state. To this end, hepatic concentration and serum levels of triiodothyronine were measured in postpubertal rats (60 days old) and adult rats (180 days old). In addition, in these rats we measured oxidative phosphorylation in homogenate together with coupled and uncoupled respiration in isolated mitochondria using succinate or durohydroquinone as substrate. We found that mitochondria from adult rats consumed less oxygen compared to younger rats due to lower electron transport chain and phosphorylating system activity. In addition, we found that in state 4 condition, mitochondria from adult rats consumed less oxygen than mitochondria from young rats. Finally, we found a decrease in liver triiodothyronine concentration in adult rats. In conclusion, the results of this study show that hepatic mitochondria in adult rats have a decreased ATP synthesis capacity and proton permeability, both consistent with the tissue hypothyroidism found in the liver of adult rats.

Citrate and succinate transport in proximal tubule cells.

Urinary citrate, which inhibits calcium nephrolithiasis, is determined by proximal reabsorption via an apical dicarboxylate transporter. Citrate is predominantly trivalent at physiological pH, but citrate(-2) is transported at the apical membrane. We now demonstrate that low-Ca solutions induce transport of citrate(-2) and succinate in opossum kidney cells. With 1.2 mM extracellular Ca, citrate uptake was pH insensitive and not competed by succinate(-2). In contrast, with low extracellular Ca, citrate uptake increased twofold, was inhibited by succinate (and other dicarboxylates), was stimulated by lowering extracellular pH (consistent with citrate(-2) transport), and increased further by lowering extracellular Mg. The effect of Ca was incrementally concentration dependent, between 0 and 1.2 mM. The effect of Ca was not simply complexation with citrate because succinate (which is complexed significantly less) was affected by Ca similarly. Incubation of cells for 48 h in a low-pH media increased citrate transport (studied at control pH) more than twofold, suggesting induction of transporters.

Demonstration of a Na(+)-dicarboxylate cotransporter in bovine adrenocortical cells.

Our study found the uptake of [14C]succinate into bovine adrenocortical cells to be sodium-dependent, inhibited by lithium, and to have an apparent K(m) of 146 mumol/l. Succinate uptake was inhibited by glutarate, fumarate, alpha-ketoglutarate, and maleate but not by 2,3-dimethylsuccinate or cis-aconitate, specific inhibitors of the basolateral Na(+)-dicarboxylate transporter of renal proximal tubule cells. Succinate uptake was highest at pH 6.0 and decreased with increasing pH. Transport of succinate was not significantly inhibited by citrate at pH 7.4 whereas at pH 6.0 inhibition of succinate uptake by citrate was small but significant. The affinity of the adrenal dicarboxylate transporter towards succinate ranges in between the low affinity of the renal luminal dicarboxylate transporter and the high affinity of the respective basolateral transporter. The pH dependency of succinate uptake and the missing inhibition by citrate at pH 7.4 differ from both the luminal and from the basolateral dicarboxylate transporters in kidney, liver, intestine, and placenta. These functional characteristics provide evidence for the existence of a Na(+)-dicarboxylate cotransporter in adrenocortical cells which may supply cholesterol metabolism with reducing substrates.