Pharmacokinetics of pioglitazone in lean and obese cats.

Pioglitazone is a thiazolidinedione insulin sensitizer that has shown efficacy in Type 2 diabetes and nonalcoholic fatty liver disease in humans. It may be useful for treatment of similar conditions in cats. The purpose of this study was to investigate the pharmacokinetics of pioglitazone in lean and obese cats, to provide a foundation for assessment of its effects on insulin sensitivity and lipid metabolism. Pioglitazone was administered intravenously (median 0.2 mg/kg) or orally (3 mg/kg) to 6 healthy lean (3.96 ± 0.56 kg) and 6 obese (6.43 ± 0.48 kg) cats, in a two by two Latin Square design with a 4-week washout period. Blood samples were collected over 24 h, and pioglitazone concentrations were measured via a validated high-performance liquid chromatography assay. Pharmacokinetic parameters were determined using two-compartmental analysis for IV data and noncompartmental analysis for oral data. After oral administration, mean bioavailability was 55%, t(1/2) was 3.5 h, T(max) was 3.6 h, C(max) was 2131 ng/mL, and AUC(0-∞) was 15 556 ng/mL · h. There were no statistically significant differences in pharmacokinetic parameters between lean and obese cats following either oral or intravenous administration. Systemic exposure to pioglitazone in cats after a 3 mg/kg oral dose approximates that observed in humans with therapeutic doses.

Evaluation of potassium iodide (KI) and ammonium perchlorate (NH4ClO4) to ameliorate 131I- exposure in the rat.

Nuclear reactor accidents and the threat of nuclear terrorism have heightened the concern for adverse health risks associated with radiation poisoning. Potassium iodide (KI) is the only pharmaceutical intervention that is currently approved by the Food and Drug Administration for treating (131)I(-) exposure, a common radioactive fission product. Though effective, KI administration needs to occur prior to or as soon as possible (within a few hours) after radioactive exposure to maximize the radioprotective benefits of KI. During the Chernobyl nuclear reactor accident, KI was not administered soon enough after radiation poisoning occurred to thousands of people. The delay in administration of KI resulted in an increased incidence of childhood thyroid cancer. Perchlorate (ClO(4)(-)) was suggested as another pharmaceutical radioprotectant for 131I- poisoning because of its ability to block thyroidal uptake of iodide and discharge free iodide from the thyroid gland. The objective of this study was to compare the ability of KI and ammonium perchlorate to reduce thyroid gland exposure to radioactive iodide (131I-). Rats were dosed with 131I- tracer and 0.5 and 3 h later dosed orally with 30 mg/kg of either ammonium perchlorate or KI. Compared to controls, both anion treatments reduced thyroid gland exposure to 131I- equally, with a reduction ranging from 65 to 77%. Ammonium perchlorate was more effective than stable iodide for whole-body radioprotectant effectiveness. KI-treated animals excreted only 30% of the (131)I(-) in urine after 15 h, compared to 47% in ammonium perchlorate-treated rats. Taken together, data suggest that KI and ammonium perchlorate are both able to reduce thyroid gland exposure to 131I- up to 3 h after exposure to 131I-. Ammonium perchlorate may offer an advantage over KI because of its ability to clear 131I- from the body.

Detection, quantifications and pharmacokinetics of toltrazuril sulfone (Ponazuril) in cattle.

Toltrazuril sulfone (Ponazuril) is a triazine-based anti-protozoal agent with highly specific actions against apicomplexan group of organisms, which are undergoing intensive investigation. Toltrazuril sulfone may have clinical application in the treatment of Neospora. caninum and other protozoal infections in cattle. To evaluate absorption, distribution, and elimination characteristics of toltrazuril sulfone in cattle, a sensitive validated quantitative high-pressure liquid chromatography method for toltrazuril sulfone in bovine biological fluids was developed. After a single oral dose of toltrazuril sulfone at 5 mg/kg (as 150 mg/g of Marquis; Bayer HealthCare, Shawnee Mission, KS, USA), samples from six cows showed good plasma concentrations of toltrazuril sulfone, which peaked at 4821 ng/mL +/- 916 (SD) at 48 h postadministration. Thereafter, plasma concentration declined to 1950 ng/mL +/- 184 (SD) at 192 h after administration with an average plasma elimination half-life of approximately 58 h. Following oral dose of toltrazuril sulfone, the observed peak plasma concentrations were in relatively close agreement ranging from the lowest 3925 ng/mL to the highest of 6285 ng/mL with the mean peak plasma concentration being 4821 ng/mL. This study shows that toltrazuril sulfone is relatively well absorbed after oral dose in cattle. These results are therefore entirely consistent with and support the reported clinical efficacy of toltrazuril sulfone in the treatment of experimentally induced clinical cases of N. caninum and other protozoal-mediated bovine diseases.

Contribution of DNA methylation to the expression of FCGRT in human liver and myocardium.

FcRn mediates recycling and transcytosis of IgG and albumin in various cell types. The MHC-class-I-like protein of the FcRn heterodimer is encoded by FCGRT. Few determinants of variable FCGRT expression in humans have been identified so far. In this study, we investigated the presence of DNA methylation in regulatory regions of FCGRT in samples of human liver and myocardium tissue, and we examined the impact of FCGRT methylation on FcRn expression in model cell lines. Quantitative DNA methylation analysis of the FCGRT locus revealed differentially methylated regions in DNA from liver and myocardium. Methylation status in individual CpG sites correlated with FCGRT mRNA expression. Data from model cell lines suggest that differential methylation in the -1058 to -587 bp regulatory region of FCGRT contributes to FcRn expression. Chromatin immunoprecipitation assays indicate that CpG site methylation impacts the binding of the methylation sensitive transcription factors Zbtb7a and Sp1. This study provides a foundation to further define the contribution of epigenetic factors during the control of FcRn expression and IgG traffic in human tissues.

Triiodothyronine differentially regulates key metabolic factors in lean and obese cats.

The effect of a 2-week administration of 75microg triiodothyronine (T3) on substrate oxidation, heat production, non-esterified fatty acids, and leptin was evaluated in eight lean (three females and five males) and eight obese (five females and three males) age-matched adult neutered cats. In addition, using real-time RT-PCR, expression of muscle and adipose tissue uncoupling proteins (UCP2 and UCP3), deiodinase 1 and 2 (D1; D2), and peroxisome proliferator-activated receptor (PPAR) alpha and gamma and peroxisome-proliferator-activator receptor-gamma co-activator 1alpha (PGC1) was examined. Compared to lean cats, obese cats had increased NEFA, leptin, UCP2, and D1mRNA in muscle and UCP3mRNA levels in fat, but lower heat production, and fat PPARs and PGC1. T3 administration increased thermogenesis and NEFA in lean and obese cats, and adipose tissue PPARgamma in lean cats. It also increased muscle D1 in lean and D2 in obese cats. The increase in muscle D2 was interpreted to be reflective of the reduced serum total T4 concentration following T3 suppression of the pituitary. No effect was seen on leptin, or UCP2 and 3. This shows that T3 regulates thermogenesis but not through changes in uncoupling protein expression. It also indicates that PPARs have an important role in the pathogenesis of obesity in cats.

Development of a feline proinsulin immunoradiometric assay and a feline proinsulin enzyme-linked immunosorbent assay (ELISA): a novel application to examine beta cell function in cats.

The prevalence of feline diabetes mellitus has increased several-fold over the last three decades. In humans, progression from obesity to diabetes is marked by changes in the release of proinsulin. A specific proinsulin (FPI) assay has not been available to examine similar changes in cats. The goal of this study was to develop a proinsulin assay for the analysis of beta cell function in cats. Monoclonal antibodies were developed against recombinant FPI and used in a two-site sandwich immunoradiometric assay (IRMA) and enzyme-linked immunosorbent Assay (ELISA). The antibody pair had negligible cross-reactivity with bovine insulin and feline C-peptide. The working range was 11-667pmol/L for the IRMA and 11-1111pmol/L for the ELISA. An intravenous glucose tolerance test was performed in six long-term obese and six lean adult healthy cats and serum glucose, insulin, and FPI concentrations were determined. The proinsulin and insulin secretion pattern in response to glucose was significantly different between lean and obese cats but the pattern was similar within a group. Both groups had similar baseline proinsulin/insulin ratios; however, obese cats showed a significantly higher proinsulin/insulin ratio during the first 15min of the IVGTT and a much lower ratio during the last 30min suggesting a time-delayed adjustment to the increased insulin demand. In conclusion, we report the development and validation of an IRMA and an ELISA for FPI. This novel assay is useful to elucidate FPI secretion and can be used similar to a C-petide assay to evaluate residual beta cell function in cats.

Regulation of the conversion of thyroxine to triiodothyronine in the perfused rat liver.

This study was undertaken to determine what factors control the conversion of thyroxine (T(4)) to triiodothyronine (T(3)) in rat liver under conditions approximating those found in vivo. Conversion of T(4) to T(3) was studied in the isolated perfused rat liver, a preparation in which the cellular and structural integrity is maintained and that can perform most of the physiologic functions of the liver. The perfused liver readily extracted T(4) from perfusion medium and converted it to T(3). Production of T(3) by the perfused liver was a function of the size of the liver, the uptake of T(4) by the liver, and the presence of T(4)-5'-deiodinase activity. Production of T(3) was increased by increasing the uptake of T(4) by liver, which could be accomplished by increasing the liver size, by increasing the perfusate T(4) concentration, or by decreasing the perfusate albumin concentration. These changes occurred without altering the conversion of T(4) to T(3). The liver had a large capacity for extracting T(4) and for T(4)-5'-deiodination to T(3), which was not saturated at a T(4) concentration of 60 mug/dl. Production of T(3) was decreased by inhibiting hepatic T(4)-5'-deiodinase with propylthiouracil, which decreased T(3) production by decreasing the conversion of T(4) to T(3). Propylthiouracil did not alter hepatic T(4) uptake. Fasting resulted in a progressive decrease in hepatic T(4) uptake to 42% of control levels by the 3rd d of fasting; this was accompanied by a proportionate decrease in T(3) production. The rate of conversion of T(4) to T(3) did not change during fasting. When T(4) uptake in 2-d-fasted rat livers was raised to levels found in fed rats by increasing the perfusate T(4) concentration from 10 to 30 mug/dl, T(3) production returned to normal. Again, no change in the rate of conversion of T(4) to T(3) was observed. These results indicate that the decreased hepatic T(3) production during fasting primarily results from decreased hepatic uptake of T(4), rather than from changes in T(4)-5'-deiodinase activity. Thus, these studies have delineated a new mechanism that functions independently of enzyme quantity or activity whereby production of T(3) from T(4) is regulated.

Obesity increases free thyroxine proportionally to nonesterified fatty acid concentrations in adult neutered female cats.

The obese cat is a model for the study of the progression toward type 2 diabetes. In this study, the impact of obesity on the hypothalamic-pituitary-thyroid axis was examined in 21 domestic shorthair cats before and after the development of obesity, which significantly increased body mass index (BMI), % body fat (BF), and girth (P<0.0001 for all). Serum total thyroxine (TT(4)), tri-iodothyronine, free T(4) (FT(4)) by direct dialysis, nonesterified fatty acids (NEFA), and leptin were measured, and FT(4) fraction (FFT(4)) was calculated. Serum thyrotropin (TSH) concentrations were measured in nine animals by validating a heterologous canine TSH assay with recombinant feline TSH as a standard. FT(4), FFT(4), NEFAs, and leptin were significantly higher in obese cats. FT(4) had the strongest positive correlation with obesity indices BF, BMI, girth, NEFA, and leptin. Fatty acids oleate and palmitate were shown to inhibit T(4) binding to pooled cat serum in vitro, suggesting the possibility that this mechanism was also relevant in vivo. Serum TT(4) and TSH did not rise significantly. The implications for thyroid hormone (TH) action are not yet clear, but fatty acids have been proposed to inhibit the cellular uptake of TH and/or pituitary TH receptor binding, leading to TH resistance. Increased leptin may also alter sensitivity to negative feedback of TH. In conclusion, feline obesity is associated with a significant increase in FT(4) within the normal range; future investigation into the cellular thyroid status will be necessary to establish cause and effect in this obesity model.

Fatty acid turnover, substrate oxidation, and heat production in lean and obese cats during the euglycemic hyperinsulinemic clamp.

Simultaneous application of the euglycemic hyperinsulinemic clamp (EHC) and indirect calorimetry was used to examine heat production, fat, and glucose metabolism in lean and obese adult neutered male and female cats. The results show that in lean insulin-sensitive cats glucose oxidation predominated during fasting, whereas lipid oxidation became more prominent in obese cats. Insulin infusion during the EHC in lean cats and obese male cats led to a large increase in glucose oxidation, glycogenesis, and lipogenesis. It also led to an increase in glucose oxidation and glycogenesis in obese female cats but it was significantly less compared to lean cats and obese males. This indicates that obese females show greater metabolic inflexibility. In obese cats of either gender, insulin caused greater suppression of non-esterified fatty acids compared to lean cats suggesting that obese cats show greater fatty acid clearance than lean cats. The heat production per metabolic size was lower in obese cats than lean cats. This would perpetuate obesity unless food intake is decreased. The higher glucose oxidation rate in obese neutered male cats suggests that they are able to replete their glycogen and lipid stores at a faster rate than females in response to insulin and it implies that they gain weight more rapidly. Further studies are needed to investigate if the different response to insulin of male cats is involved in their higher risk to develop diabetes.

Cloning and sequencing of feline thyrotropin (fTSH): heterodimeric and yoked constructs.

The genes encoding the mature common glycoprotein alpha (CGA) and hormone-specific beta subunits of feline thyroid stimulating hormone (fTSH) were cloned and sequenced. The feline CGA gene was cloned from RNA extracted from the feline pituitary gland by the reverse transcription polymerase chain reaction (RT-PCR). The gene fragment that encodes mature TSHbeta was cloned from feline genomic DNA after direct polymerase chain reaction (PCR). In both cases, primers were based on the consensus sequences from TSH in other species. The resulting 510 bp PCR product for the CGA-subunit included the full coding sequence for the 96 amino acid mature subunit preceded by a 24 amino acid signal peptide. The 850 bp sequence of fTSHbeta genomic DNA consisted of two coding exons, an intron of 418 bp, and a 60 bp signal sequence. The octapeptide immunoaffinity tag FLAG was added to 3' end of the alpha gene to facilitate detection and purification. Both genes were cloned independently downstream from the EF1alpha promoter of the PEAK transfer vector to facilitate co-expression studies in PEAK cells (modified human embryonic kidney (HEK) cells). A single-chain analogue of fTSH termed yoked fTSH (yfTSH) was developed by fusing the nucleotides encoding the C-terminus of the beta-subunit fused to the N-terminus of the alpha-subunit with DNA encoding the C-terminal peptide (CTP) of human chorionic gonadotropin beta-subunit as a linker peptide. The resulting single-chain analogue encoded from N-terminus to C-terminus: beta-CTP-alpha-FLAG. The resulting DNA sequence was cloned, sequenced, ligated and recloned into expression vector PEAK. This report constitutes the first cloning and sequencing of the genes encoding the subunits of feline thyrotropin.

Assessment and mathematical modeling of glucose turnover and insulin sensitivity in lean and obese cats.

Insulin sensitivity (SI) of glucose disposal can be quantified with the euglycemic hyperinsulinemic clamp (EHC) with tracer glucose infusion. True steady state is, however, difficult to achieve, and non-steady state analysis of EHC data is preferred. This analysis requires information on glucose kinetics that can be obtained from bolus injection of cold and tracer glucose. The aim of this study was to assess glucose kinetics in cats. Mathematical modeling and non-steady state analysis was applied to assess effects of obesity on glucose turnover, glycolysis/glycogen synthesis, SI, and inhibition of endogenous glucose production (EGP) in lean cats (L) and obese cats (O). D-[3-(3)H]-glucose kinetics and 3H-H2O production were analyzed in 4 L and 4 O with three-compartment modeling. Frequently sampled insulin-modified intravenous glucose tolerance tests (FSIGT) with minimal model analysis were performed in 5L and 3 O to assess glucose kinetics and SI. EHC was performed in 10 L and 10 O with primed-constant infusion of 3H-glucose. Data were analyzed with a modified minimal model segregating suppression of EGP by insulin using a non-linear mixed-effects population approach. FSIGT provided estimates of SI, glucose effectiveness SG, and distribution volume. EHC provided estimates of SI, SG, glycolysis, and suprabasal insulin concentration for 50% EGP inhibition. Obesity appears to affect glucose distribution but not utilization at basal insulin, and reduces SI estimated by FSIGT and EHC. Differences in SI between FSIGT and EHC depend on different descriptions of EGP inhibition by insulin. Finally, glucose disposal at basal insulin appears to occur entirely through glycolysis, whereas significant amounts of glucose are sequestrated from oxidation during EHC.

Cloning, expression and purification of feline proinsulin.

Feline proinsulin was cloned and expressed using a bacterial expression system. It was then purified from inclusion bodies using size exclusion chromatography and further processed including reduction of the protein. Following refolding, proinsulin was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). RP-HPLC and mass spectrometric analysis indicated that the proinsulin contained the correct disulfide bridging pattern. This proinsulin can be used for therapeutic and diagnostic purposes.

Expression and purification of feline thyrotropin (fTSH): immunological detection and bioactivity of heterodimeric and yoked glycoproteins.

The goal of this study was to express and purify recombinant feline TSH as a possible immunoassay standard or pharmaceutical agent. Previously cloned feline common glycoprotein alpha (CGA) and beta subunits were ligated into the mammalian expression vector pEAK10. The feline CGA-FLAG and beta subunits were cloned separately into the pEAK10 expression vector, and transiently co-transfected into PEAK cells. Similarly, previously cloned and sequenced yoked (single chain) fTSH (yfTSH) and the CGA-FLAG sequences were ligated into the same vector, and stable cell lines selected by puromycin resistance. Expression levels of at least 1 microg/ml were achieved for both heterodimeric and yoked fTSH forms. The glycoproteins were purified in one step using anti-FLAG immunoaffinity column chromatography to high purity. The molecular weights of feline CGA-FLAG subunit, beta subunit and yfTSH were 20.4, 17, and 45 kDa, respectively. Both heterodimeric and yoked glycoproteins were recognized with approximately 40% detection by both a commercial canine TSH immunoassay and an in-house canine TSH ELISA. The yoked glycoprotein exhibited parallelism with the heterodimeric form in the in-house ELISA, supporting their possible use as immunoassay standards. In bioactivity assays, the heterodimeric and yoked forms of fTSH were 12.5 and 3.4% as potent as pituitary source bovine TSH at displacing (125)I-bTSH and 45 and 24% as potent in stimulating adenylate cyclase activity in human TSH receptor-expressing JP09 cells. However, in addition to reduced receptor binding affinity, the recombinant glycohormones produced a reduced maximal effect at maximal concentration (E(max)) suggesting the possibility of the recombinant glycohormone constructs acting as partial agonists at the human TSH receptor.

Activity and tissue-specific expression of lipases and tumor-necrosis factor alpha in lean and obese cats.

Post-heparin plasma activity of lipoprotein lipase (LPL) and hepatic lipase (HL), and fat and muscle activity of LPL were measured in neutered lean and obese cats. Lipoprotein lipase, hormone-sensitive lipase (HSL), and tumor necrosis factor a (TNF) mRNA were measured in muscle and fat tissue with real-time PCR using primers for feline LPL, HSL, and TNF. Lipoprotein lipase plasma and fat activity and fat mRNA levels were significantly lower (50, 80, and 50%, respectively) in obese cats than lean cats, whereas the muscle/fat ratio of LPL was significantly higher in obese compared to lean cats. The activity of HL was not different between the groups. Hormone-sensitive lipase mRNA levels were significantly higher in obese than lean cats. The level of fat TNF also was significantly higher in obese cats than in lean cats, whereas the level in muscle was not different. The lower LPL activity and mRNA expression in fat and the higher LPL and HSL mRNA expression in muscle in obese cats compared to lean cats expectedly favor a redistribution of fatty acids from fat to muscle tissue where they can be deposited or used for energy in times of need. Tumor necrosis factor alpha may regulate this repartitioning process through suppression of adipocyte LPL.

Glucose inhibits the high-affinity (Ca2+ + Mg2+)-ATPase in the plasma membrane of a glucose-responsive insulinoma.

(Ca2+ + Mg2+)-ATPase enzyme activity of a purified plasma membrane preparation from a glucose responsive rat insulinoma, was characterized as Ca2(+)-dependent dephosphorylation of [gamma-32P]ATP. A high-affinity enzyme with a Km(ATP) ranging from 20 to 30 microM and a Km(Ca2+) of 1 microM was identified. Glucose inhibited this high-affinity enzyme in a dose-dependent manner, with no significant inhibition at a concentration between 0 and 5 mM, 50% inhibition at 13.3 mM and 94.5% inhibition at 30 mM. The inhibitory effect of glucose was immediate and rapidly reversible. The effect was stereospecific for the alpha-anomer. These findings support the concept that glucose acts directly at the beta-cell plasma membrane and is involved in the maintenance of elevated intracellular free calcium concentrations associated with insulin release by directly or indirectly inhibiting energy-dependent calcium efflux. Glyceraldehyde (20 mM) increased enzyme activity 3-fold, while other metabolic fuels had no effect. This suggests that inhibition of the enzyme is not an obligatory requirement for insulin release. Calmodulin stimulated the enzyme activity in calmodulin-depleted but not in undepleted membranes. Trifluoperazine (30-100 microM) inhibited (Ca2+ + Mg2+)-ATPase in a dose-dependent manner (14-61% activity) and the activity was also inhibited by vanadate (0.1-1.0 mM) and NaCl (150 mM).

Fuel-induced insulin release in vitro from insulinomas transplanted into the rat kidney.

We studied the release of insulin, glucagon, and somatostatin in response to glucose, glyceraldehyde (GA), and alpha-ketoisocaproate (KIC) from rat kidneys containing transplanted insulinomas. Kidneys were perfused about 11 wk after transplantation when the plasma glucose concentration of the fed animals had decreased from 180 +/- 7 to 95.1 +/- 9.9 mg/dl and plasma insulin concentrations had increased from 2.6 +/- 0.5 to 14.2 +/- 2.0 ng/ml. The insulin content of the tumor-containing kidney ranged from 40 to 679 micrograms; the glucagon and somatostatin concentrations ranged from undetectable levels to 3.7 micrograms and 248 ng, respectively. The average response to 30 mM glucose and 10 mM GA was a four- to fivefold increase in insulin secretion, whereas 30 mM KIC caused a 16- to 28-fold increase. In vitro stimulation of the insulinoma with 30 mM glucose primed the beta-cell response to a second stimulus following a short rest period. Cytochalasin B did not enhance this primed glucose response. Diazoxide inhibited glucose, GA, and KIC-stimulated insulin release. Glucose, GA, and KIC stimulated glucagon release in 2 of 17 insulinomas studied here. Somatostatin release was not seen in any of the experiments. These findings show that this islet cell tumor transplanted under the kidney capsule releases insulin in response to physiologic and model fuel substances. Thus, this particular transplantable tumor offers an opportunity to study the biochemistry and biophysics that underlie fuel-stimulated insulin release.

Structure-based design of a constrained peptide mimic of the HIV-1 V3 loop neutralization site.

Antigenic variation among different HIV-1 isolates has been a major problem in the development of an effective vaccine against AIDS. Peptide vaccines incorporating structural elements common to groups of viral isolates, such as the clade subtypes of HIV-1, hold promise; however, the design of such immunogens has been hampered by the lack of specific structural information on the viral proteins to be targeted. As part of a structure-based approach to this problem, we report the design and characterization of a conformationally restricted peptide analog (Aib142) of a highly conserved HIV-1 clade-B sequence from the third variable loop of the membrane glycoprotein gp120. The design strategy incorporates peptide conformational data derived from crystal structure analysis of an MN-isolate peptide (RP142) in complex with the Fab fragment (Fab59.1) of a broadly neutralizing antibody. The synthetic peptide (Aib142) replaces an alanine residue within the V3 loop epitope sequence GPGRAF by the conformationally restricted helicogenic alpha-aminoisobutyryl residue. As expected, the crystal structure of the Fab 59.1-Aib142 complex at 2.8 A resolution shows that the peptide interacts very similarly with the neutralizing antibody. Proton nuclear magnetic resonance (NMR) studies indicate that the free Aib142 peptide is indeed more ordered in solution with a conformational preference that corresponds to the X-ray structure of its Fab-bound form. Aib142 thus represents the first step in the design of conformationally constrained peptide analogs built to mimic biologically relevant structural forms of HIV-1 neutralization sites.

Na+/Ca2+ exchange in plasma membrane vesicles from a glucose-responsive insulinoma.

Plasma membrane vesicles from a glucose-responsive insulinoma exhibited properties consistent with the presence of a membrane Na+/Ca2+ exchange. The exchange was rapid, reversible, and was dependent on the external Ca2+ concentration (Km = 4.1 +/- 1.1 microM). External Na+ inhibited the uptake in a dose-dependent manner (IC50 = 15 mM). Dissipation of the Na+ gradient by 10 microM monensin decreased Na+/Ca2+ exchange from 0.74 +/- 0.17 nmoles/mg protein/s to 0.11 +/- 0.05 nmoles/mg protein/s. Exchange was not influenced by veratridine, tetrodotoxin and ouabain, or by modifiers of cAMP. No effect was seen using the calcium channel blockers, nitrendipine or nifedipine. Glucose had no direct effect on Na+/Ca2+ exchange, while glyceraldehyde, glyceraldehyde-3-phosphate and dihydroxyacetone inhibited the exchange. Na+ induced efflux of calcium was seen in Ca2+ loaded vesicles and was half maximal at [Na+] of 11.1 +/- 0.75 mM. Ca2+ efflux was dependent on [Na+], with a Hill coefficient of 2.7 +/- 0.07 indicating that activation of Ca2+ release involves a minimum of three sites. The electrogenicity of this exchange was demonstrated using the lipophilic cation tetraphenylphosphonium [( 3H]-TPP), a membrane potential sensitive probe. [3H]-TPP uptake increased transiently during Na+/Ca2+ exchange indicating that the exchange generated a membrane potential. These results show that Na+/Ca2+ exchange operates in the beta cell and may be an important regulator of intracellular free Ca2+ concentrations.

Kinetics of triiodothyronine dissociation from bovine serum albumin: modification of the resin capture method with subsequent computer modeling.

There has been much debate about the mechanism by which thyroid hormone leaves the circulation and enters cells. Critical to the development of theoretical models of thyroid hormone uptake are precise estimates of the kinetic constants describing the reversible binding of the hormone to its binding proteins. A resin capture method was modified to permit the collection of dissociation data at intervals of 0.12 sec. Columns (30 X 0.6 cm) of Amberlite IRA 400 anion exchange resin were perfused at 30 ml/min with 3.5% dextran in modified Krebs Henseleit bicarbonate buffer at pH 7.4 and 37 C. Injection of [125I]triiodothyronine (T3) tracer in BSA-free medium established the rate constant for resin capture of free T3 in this system: kRes = 0.89 +/- 0.04 sec-1 (mean +/- SD, n = 4). [125I]T3 preequilibrated with BSA was infused to achieve final BSA concentrations of 2.3, 48, 120, 290, 507, and 725 microM, yielding apparent uptake rate constants (kapp) of 0.77 +/- 0.12 (n = 6), 0.23 +/- 0.01 (n = 4), 0.17 +/- 0.04 (n = 4), 0.057 +/- 0.005 (n = 4), 0.039 +/- 0.003 (n = 4), 0.020 +/- 0.002 (n = 4) sec-1 (SSE less than 9.4 X 10(-2), respectively, when fitted by nonlinear methods. Solving the equation kapp = kRes/(1 + [BSA]/KD') gave KD' = 1.80 +/- 0.07 X 10(-5) M (r2 = 0.96, P less than 0.001, n = 26) where KD' is the apparent or flow equilibrium constant. In a further experiment T3:BSA reassociation was eliminated by BSA dilution. This resulted in determination of the dissociation rate constant kdissoc = 0.63 +/- 0.16 sec-1 (n = 8) and therefore, the association rate constant kassoc = 3.5 X 10(4) M-1 sec-1. Mathematical modeling predicted that 14.4 microM BSA would decrease the rate of T3 uptake by the resin system by 65%. The free fraction of T3 during a single pass extraction was predicted to be greater than that measured by equilibrium dialysis, suggesting that dissociation of T3 from BSA is favored in the flux model relative to that measured at equilibrium in vitro.

Effect of dexamethasone on triiodothyronine production in the perfused rat liver and kidney.

Dexamethasone administration to rats decreases T4-5'-deiodinase activity in liver homogenates and slices and in isolated rat renal tubules. To determine if this decreased T4-5'-deiodinase activity results in decreased T3 production, rat livers and kidneys of control and dexamethasone-treated rats were perfused with medium containing free T4 concentrations approximating euthyroid rat serum, and net T3 production was measured by RIA. Dexamethasone administration decreased body weight by 14% but did not affect liver weight, kidney weight, or serum concentrations of T4 or T3. When livers were perfused with T4 concentrations of 10 micrograms/dl (free T4 = 6.5 ng/dl), hepatic T3 production, T4 uptake, and the conversion of T4 to T3 were similar in dexamethasone-treated rats and saline-treated controls. However, when livers were perfused at a T4 concentration of 125 micrograms/dl (free T4 = 81 ng/dl), dexamethasone-treated livers produced significantly less T3 than controls because of decreased conversion of T4 to T3. Hepatic deiodination of T3 and excretion of T3 into bile were not affected by dexamethasone. Renal T3 production, T4 uptake, and conversion of T4 to T3 was likewise unaffected by dexamethasone treatment when kidneys were perfused at near-normal free T4 concentrations. These studies indicate that dexamethasone treatment does not alter T3 production in the perfused liver and kidney and underscore the importance of using free T4 concentrations approximating physiologic levels when studying regulation of T3 production in individual organs.