Intravenous injection of human sex steroid hormone-binding globulin in mouse decreases blood clearance rate and testicular accumulation of orally administered [2-125I]iodobisphenol A

Bisphenol A, an environmental compound with estrogenic activity, has been shown to bind human sex steroid hormone-binding globulin (hSHBG), the main plasma transport protein which regulates the metabolism of androgens and estrogens and limits their access to target organs. The present study was conducted to determine whether physiologically relevant concentrations of hSHBG can influence the blood clearance rate of bisphenol A and its accumulation in the testes. A radioactive [2-125I]iodobisphenol tracer was synthesized with an association constant (Ka) for binding to hSHBG of 0.14 +/- 0.01 x 10(6) M(-1) at 37 degrees C, a value much lower than for [2-125I]iodoestradiol, which was also synthesized. We used i.v. injection of immunopurified hSHBG in adult male mice to maintain hSHBG levels within the physiologically possible range for humans (27-267 nM) before gavage administration of [2-125I]iodobisphenol or [2-125I]iodoestradiol, for measuring the blood clearance rate of radioactive signal in blood samples taken during the following 120 min. Testicular accumulation of radioactivity was measured 24 h and 48 h after gavage of [2-125]iodobisphenol A. In mice receiving immunopurified hSHBG or vehicle, the time-dependent blood clearance of radioactivity exhibited a bi-exponential decrease which indicated alpha-diffusion and beta-elimination phases for both radioactive ligands. The presence of circulating hSHBG significantly and dose-dependently lowered the clearance rate of radioactivity. However, much higher circulating levels of hSHBG were required to retard the blood clearance of [2-125I]iodobisphenol A as compared to those required for [2-125I]iodoestradiol, in keeping with the important difference in their respective Ka value for binding to SHBG. In addition, mice treated with hSHBG exhibited significantly (P = 0.036) reduced testicular accumulation of radioactivity 24 h and 48 h after ingestion of [2-125I]iodobisphenol A. Provided that the binding properties of bisphenol A for hSHBG are not substantially different from those measured for [2-125I]iodobisphenol A, these findings suggest that, although hSHBG binds 2-mono-iodobisphenol A with a relatively low binding affinity, high enough concentrations of circulating hSHBG (range concentrations between 85 and 267 nM) are potentially able to exert a protective effect against exposure to bisphenol A.     

Progesterone regulation of vascular thromboxane A(2) receptors in rhesus monkeys

We hypothesized that progesterone regulates thromboxane A(2) receptor (TxA(2)R) density in primate vascular muscle and that TxA(2)R density correlates with coronary reactivity in vivo and in vitro. Reactivity to serotonin + U-46619 was determined by angiography in surgically postmenopausal [ovariectomized (Ovx)] rhesus monkeys without progesterone replacement and after 2-wk progesterone treatment (1-2 ng/ml). In untreated Ovx animals, 100 micromol/l serotonin + 1 micromol/l U-46619 (syringe concentrations) provoked vasospasm-like constrictions in six of six monkeys; zero of six progesterone-treated monkeys developed vasospasms. Sustained Ca(2+) responses in vascular muscle cells isolated from Ovx coronaries (208 +/- 63% of basal 20 min after stimulation) treated with serotonin + U-46619 contrasted with transient Ca(2+) responses (143 +/- 18% of basal and decreasing 5 min after stimulation) in progesterone-treated monkeys. The maximum density of [1S-(1I,2J(5Z),3I(1E,3R*),4I)]-7-[3-(3-hydroxy-4-(4'-[(125)I]iodophenoxy)- 1-butenyl)-7-oxabicyclo[2.2.1]heptan-2-yl]-5-heptenoic acid ([(125)I]-BOP) binding was greater (P < 0.01) in carotid arteries and aortic membranes from Ovx (109 +/- 11 fmol/mg) compared with progesterone-treated (43 +/- 15 fmol/mg) monkeys. TxA(2)R immunolabeling revealed greater coronary TxA(2)R labeling in Ovx compared with progesterone-treated monkeys. The results suggest that progesterone can decrease arterial TxA(2)R in Ovx monkeys.     

The effects of sex hormone binding globulin (SHBG) on testosterone transport into the cerebrospinal fluid.

The movement of testosterone (T) from blood across the blood-brain barrier (BBB) is thought to reflect the combined effects of T's lipid solubility and the presence of circulating binding proteins for T such as albumin or sex hormone binding globulin (SHBG). Since the adult rat lacks a circulating specific high affinity sex steroid binding protein, examination of the disappearance from serum and uptake into cerebrospinal fluid (CSF) of [3H]T before and after SHBG or albumin infusion should provide insight into the function of these two proteins with respect T transport. Three groups of adult male Sprague-Dawley rats were cannulated at the femoral vein and cisterna magna. In a control group (n = 8), [3H]T was given as an intravenous bolus beginning at time zero; multiple serum and CSF collections were assayed for counts per min (cpm) during the subsequent 45 min. Data from these animals were then compared to those seen in animals that received either purified human SHBG (hSHBG) (n = 7) or human albumin (hALB) (n = 6) 10 min prior to the [3H]T infusion. High performance liquid chromatography was used to monitor the metabolic fate of the steroid infusate at the end of each study period. Infusion of hSHBG increased serum concentrations from undetectable to 93.8 nM/l (mean +/- SEM, n = 6). Administration of hALB significantly increased (25.0 +/- 1.2 g/l at baseline, 33.4 +/- 1.6 g/l post-infusion, mean +/- SEM, P less than 0.03, n = 5) the circulating albumin concentration. Comparison of data from each group of animals demonstrated that (1) following an i.v. injection of radiolabeled T, the initial decline in serum [3H]T was significantly reduced (P less than 0.03) in the presence of hSHBG, (2) hALB did not affect the movement of [3H]T out of serum, (3) the time to peak appearance of [3H]T in the CSF was significantly delayed (P less than 0.02) by the presence of circulating hSHBG, and (4) the net quantity of [3H]T found in the cSF under steady-state conditions was not affected by serum SHBG or albumin levels. This study demonstrates that high-affinity steroid binding proteins do modulate the transport of sex steroids across the BBB. Specifically, SHBG delays the clearance of T from serum and slows the rate of T uptake into the CSF during non-equilibrium conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and Characterization of Binding of 5-[76Br] Bromo-3-[[2(S)-Azetidinyl]methoxy]pyridine, a Novel Nicotinic Acetylcholine Receptor Ligand, in Rat Brain

5-[76Br]Bromo-3-[[2(S)-azetidinyl]methoxy]pyridine ([76Br]BAP), a novel nicotinic acetylcholine receptor ligand, was synthesized using [76Br]bromide in an oxidative bromodestannylation of the corresponding trimethylstannyl compound. The radiochemical yield was 25%, and the specific radioactivity was on the order of 1 Ci/micromol. The binding properties of [76Br]BAP were characterized in vitro and in vivo in rat brain, and positron emission tomography (PET) experiments were performed in two rhesus monkeys. In association experiments on membranes of the cortex and thalamus, >90% of maximal specific [76Br]BAP binding was obtained after 60 min. The dissociation half-life of [76Br]BAP was 51 +/- 6 min in cortical membranes and 56 +/- 3 min in thalamic membranes. Saturation experiments with [76Br]BAP revealed one population of binding sites with dissociation constant (K(D)) values of 36 +/- 9 and 30 +/- 9 pM in membranes of cortex and thalamus, respectively. The maximal binding site density (Bmax) values were 90 +/- 17 and 207 +/- 33 fmol/mg in membranes of cortex and thalamus, respectively. Scatchard plots were nonlinear, and the Hill coefficients were <1, suggesting the presence of a lower-affinity binding site. In vitro autoradiography studies showed that binding of [76Br]BAP was high in the thalamus and presubiculum, moderate in the cortex and striatum, and low in the cerebellum and hippocampus. A similar pattern of [76Br]BAP accumulation was observed by ex vivo autoradiography. In vivo, binding of [76Br]BAP in whole rat brain was blocked by preinjection of (S)(-)-nicotine (0.3 mg/kg) by 27, 52, 68, and 91% at survival times of 10, 25, 40, 120, and 300 min, respectively. In a preliminary PET study in rhesus monkeys, the highest [76Br]BAP uptake was found in the thalamus, and radioactivity was displaceable by approximately 60% with cytisine and by 50% with (S)(-)-nicotine. The data of this study indicate that [76Br]BAP is a promising radioligand for the characterization of nicotinic acetylcholine receptors in vivo.     

The effects of l-thyroxine and dexamethasone on steroid dynamics in male cynomologous monkeys

Male cynomologous monkeys (M. fascicularis) were infused with [3H]androgens, [14C]estrogens and [3H]cortisol before and after the administration of l-thyroxine, (l-T4) 150 micrograms/day for 6 wk, dexamethasone 8 mg every 8 h for 3 doses and dexamethasone 1.0 mg/day for 8 days. Blood samples were obtained before each of the infusions and analyzed for endogenous T, A, E1, E2 and F concentrations, % free T and % free E2, sex hormone-binding globulin (SHBG) and cortisol binding globulin (CBG) capacity. When l-T4 was being administered, T4 and triiodothyronine (T3) concentrations were also measured. Blood samples were obtained during the infusions and analyzed for radioactivity as testosterone (T), androstenedione (A), dihydrotestosterone (DHT), estradiol (E1), estrone (E2), and cortisol (F). All urine was collected for 96 h and an aliquot of the pooled urine was analyzed for radioactivity as estrone and estradiol glucuronide. The administration of l-T4 for 6 wk to 3 monkeys resulted in a marked rise in T4 and T3 levels, from 4.8 +/- 0.4 micrograms/dl and from 136 +/- 6 to 515 +/- 71 ng/dl, respectively. MCRT, MCRE2 and MCRE1 did not change, but MCRA values increased slightly and MCRF increased 2-3 fold. [rho]T.E2 did not change but [rho]A.E1BM showed a slight but significant increase. The inter-conversions between the androgens and between the estrogens were not altered. There was a 2-3-fold increase in SHBG and a decrease in %FT but no change in %FE2 or CBG. The concentrations of T, A and DHT rose but there was no trend in the levels of the estrogens. The administration of dexamethasone 8 mg every 8 h for 3 doses or 1 mg/day for 8 days caused no changes in the MCRs for T, A, E1 and E2 but did cause a significant decrease in MCRF. Measurement of splanchnic and peripheral tissue extractions before and after acute dexamethasone administration in 1 monkey showed that the decrease in MCRF was the result of a marked decrease, 11-2%, in splanchnic extraction of F. The extractions of T and E2 were relatively unaffected. The concentrations of T and F fell but E2 remained the same. % FT and % FE2 rose slightly and the concentrations of SHBG and CBG were unchanged. The androgen interconversions and estrogen interconversions were not affected but [rho]T,E2BM and [rho]A,E1BM showed slight decreases.(ABSTRACT TRUNCATED AT 400 WORDS)     

On the enterohepatic cycle of triiodothyronine in rats; importance of the intestinal microflora

Until 70 h after a single iv injection of 10 uCi [125I]triiodothyronine (T3), normal rats excreted 15.8 +/- 2.8% of the radioactivity with the feces and 17.5 +/- 2.7% with the urine, while in intestine-decontaminated rats fecal and urinary excretion over this period amounted to 25.1 +/- 7.2% and 23.6 +/- 4.0% of administered radioactivity, respectively (mean +/- SD, n = 4). In fecal extracts of decontaminated rats 11.5 +/- 6.8% of the excreted radioactivity consisted of T3 glucuronide (T3G) and 10.9 +/- 2.8% of T3 sulfate (T3S), whereas no conjugates were detected in feces from normal rats. Until 26 h after ig administration of 10 uCi [125I]T3, integrated radioactivity in blood of decontaminated rats was 1.5 times higher than that in normal rats. However, after ig administration of 10 uCi [125I]T3G or [125I]T3S, radioactivity in blood of decontaminated rats was 4.9- and 2.8-fold lower, respectively, than in normal rats. The radioactivity in the serum of control animals was composed of T3 and iodide in proportions independent of the tracer injected, while T3 conjugates represented less than 10% of serum radioactivity. These results suggest an important role of the intestinal microflora in the enterohepatic circulation of T3 in rats.     

Effects of pleural pressure and abdominal pressure on diaphragmatic blood flow

Alveolar transfer of prostaglandin E2 (PGE2) was characterized in isolated perfused guinea pig lungs (n = 19) by measuring radioactivity appearing in the venous effluent during 30 min after intratracheal instillation of [3H]PGE2, [14C]-mannitol, and [125I]iodoantipyrine. Recovery of lipid-soluble [125I]iodoantipyrine [91 +/- 3% (SE)] after 30 min was used to estimate total 3H and 14C delivered to the exchanging region of lung at time 0. In seven control lungs, 58 +/- 4% of [14C]mannitol and 16 +/- 4% of [3H]PGE2 was retained 10 min after instillation. Neither perfusion with diphloretin phosphate (10 micrograms/ml; n = 4) nor hypothermia (5 degrees C; n = 5) significantly affected the amount of [14C]mannitol retained; however, [3H]PGE2 remaining in these lungs increased significantly to 36 +/- 4 and 53 +/- 2%, respectively. Addition of unlabeled PGE2 (200 micrograms) to the instilled solution (n = 3) increased retention of both [14C]mannitol (80 +/- 3%) and [3H]PGE2 (65 +/- 4%). Alveolar transfer of [3H]PGE2 was calculated as the difference in percent retention of [14C]mannitol and [3H]PGE2 and normalized to that of [14C]mannitol. After 10 min, alveolar transfer of [3H]PGE2 was 71 +/- 8% in control lungs but was decreased to 26 +/- 7, 10 +/- 5, and 19 +/- 6% by diphloretin phosphate, hypothermia, or unlabeled PGE2, respectively. These data suggest that alveolar clearance of PGE2 involves a saturable drug- and temperature-sensitive process.     

Synthesis and properties of sulfhydryl-group-specific reagents containing 125I.

125I-containing compounds that react specifically with sulfhydryl groups were prepared in yields of 30 to 40% on the basis of starting 125I quantity. The synthetic precursors were commercially available heterobifunctional crosslinkers and the peptide L-arginyl-L-tyrosine. Two types of sulfhydryl specific reagents were prepared: 3-(2-pyridyldithio)propionylarginyl-[125I]-monoiodotyrosine, which permits reversible incorporation of 125I at sulfhydryl sites, and 3-maleimidopropionylarginyl- [125I]monoiodotyrosine, an irreversible labeling reagent. These products were isolated in a highly radiochemically pure form by C18 HPLC. The second-order rate constants for the reaction of 3-(2-pyridyldithio)propionylarginylmonoiodotyrosine and 3-maleimidopropionylarginylmonoiodotyrosine with N-acetylcysteine were 28 +/- 3 M-1 s-1 and 154 +/- 4 M-1 s-1, respectively, at pH 7.3. Storage of carrier-free 3-(2-pyridyldithio)propionylarginyl-[125I]monoiodotyrosine and 3-maleimidopropionylarginyl-[125I]monoiodotyrosine at -80 degrees C at a radioactive concentration of 0.4 mCi/ml resulted in conversion of 125I to species that did not react covalently with sulfhydryl groups. This process occurred with first-order kinetics and a t1/2 of 5.7 days for the pyridyldithio compound and 7.5 days for the maleimido compound. No conversion was observed during storage at -80 degrees C at radioactive concentrations of 0.02 mCi/ml or less. The labeling properties of these compounds were examined using red blood cell proteins as a test system. 3-(2-Pyridyldithio)propionylarginyl- [125I]monoiodotyrosine and maleimidopropionylarginyl-[125I]monoiodotyrosine reacted preferentially with membrane - associated sulfhydryl groups when incubated with intact red blood cells.     

Studies of the penetration of the blood brain barrier by atrial natriuretic factor

The atrial natriuretic factors (ANF) have been detected in various areas of the brain. To determine whether circulating blood borne ANF could contribute to the ANF content in the central nervous systems we examined the ability of ANF-99-126 or ANF-102-126 to penetrate the blood brain barrier. Carotid artery injections of [3H] inulin with [125I] ANF in anesthetized rabbits resulted in a comparably minimal brain uptake index (BUI) for each labeled substance as measured in cerebral cortex extracts. Injection of [3H] HOH and [125I] ANF resulted in a mean BUI in cortex of 4.9 +/- .6 (SEM)% for ANF relative to triated water; this low uptake was not significantly saturable. The BUI ratio for ANF/HOH in olfactory bulb was somewhat higher though still low, at 7.0 +/- 9%, possibly reflecting the high density of ANF receptors in this structure. Infusion of [125I] ANF into the carotid artery of anesthetized rabbits resulted in little radioactivity being detected in the cerebrospinal fluid. Infusion of unlabeled ANF, which raised plasma levels as high as 26.3 ng/ml, resulted in little change in CSF levels. Our results demonstrate that the uptake of ANF into the brain is minimal and supports the idea that local synthesis of ANF predominantly accounts for the brain pool of this peptide.     

Pharmacokinetics and lung distribution of a humanized anti-RAGE antibody in wild-type and RAGE-/- mice

A neutralizing antibody to the receptor for the advanced glycation end products (anti-RAGE Ab) was developed as a potential treatment of acute and chronic inflammatory conditions. Previous pharmacology studies demonstrated efficacy of the anti-RAGE antibody in the mouse model of sepsis. We examined pharmacokinetics and lung distribution of [¹²⁵I]anti-RAGE Ab in RAGE-/- and wild-type (129S5) mice following single IV administration. Serum pharmacokinetics of [¹²⁵I]anti-RAGE Ab was similar in RAGE-/- and 129S5 mice, with the total body clearance of 0.3 mL/hr/kg and the elimination half-life of 11-12 days, suggesting the target expression had limited impact on overall elimination of [¹²⁵I]anti-RAGE Ab from mice. [¹²⁵I]Anti-RAGE Ab accumulated in the lung of 129S5 mice, with ~4% of total dose retained in the lung at days 6-27 and the lung AUC0-∞ of ~300% of that in serum. The SDS-PAGE analysis suggested that most of retained lung radioactivity was attributed to intact antibody. No accumulation of radioactivity was observed in the lung of RAGE-/- mice, indicating that lung uptake of [¹²⁵I]anti-RAGE Ab was target-dependent in wild-type mice. These data suggest that the anti-RAGE Ab was able to localize to the site of RAGE expression, the lung, and support the findings in the previous pharmacology studies.     

Estrogen metabolism as measured in blood and urine in female rhesus monkeys

In order to measure the interconversions of estrone (E1) and estradiol (E2) and their conversion to the 16 alpha-hydroxylated estrogens, 16 alpha-hydroxy estrone (16 alpha-OHE1) and estriol (E3), we infused 11 female rhesus monkeys with [3H]E2 and [14C]E1 and measured radioactivity in the blood as E1, E2 and 16 alpha-OHE1 (n = 9) and in the urine as the glucuronides of E1, E2, 16 alpha-OHE1, and E3 (n = 11). The mean conversion of E1 to E2 as measured in blood (percent of infused E1 measured in blood as E2, [rho]1.2BB) was 29.2 +/- 1.6% and as measured in the urine of the same animals, [rho]1.2BM, was 77.4 +/- 5.9%. The mean conversion of E2 to E1, [rho]2.1BB was 21.5 +/- 1.0% and as measured in urine, [rho]2.1BM was 67.7 +/- 4.6%. Thus for both estrone and estradiol only 30-35% of the interconversions occurred in pools which were in equilibrium with the blood pool of these estrogens. The remaining 65-70% occurred in a pool, probably liver, in which glucuronidation occurred immediately after conversion. The conversion ratios (the ratio of the concentration in the blood of radioactivity as 16 alpha-OHE1 to its precursor, CRPrec,16 alpha-OHE1) was 0.036 +/- 0.008 for CRE1,16 alpha-OHE1 and 0.0039 +/- 0.0010 for CRE2,16 alpha-OHE1. The percentages of administered E1 excreted in the urine as the glucuronides of E1, E2, 16 alpha-OHE1 and E3 were 20.1 +/- 1.5, 1.6 +/- 0.2, 0.96 +/- 0.20 and 0.76 +/- 0.07 respectively. The percentages of administered E2 excreted in the urine as E1, E2, 16 alpha-OHE1 and E3 were 14.4 +/- 1.0, 2.2 +/- 0.3, 0.57 +/- 0.05 and 0.68 +/- 0.11 respectively. Thus there are minor differences in the patterns of excreted metabolites of E1 and E2. Furthermore, 16 alpha-OHE1 and E3 are not major metabolites of E1 or E2 in the female rhesus monkey.     

Protein transfer between A-I-containing lipoprotein subpopulations: evidence of non-transferable A-I in particles with A-II.

Transfer of apolipoproteins (apo) between the two subpopulations of apo A-I-containing lipoproteins in human plasma: those with A-II [Lp(AI w AII)] and those without [Lp(AI w/o AII)], were studied by observing the transfer of 125I-apo from a radiolabeled subpopulation to an unlabeled subpopulation in vitro. When Lp(AI w AII) was directly radioiodinated, 50.3 +/- 7.4 and 19.5 +/- 7.7% (n = 6) of the total radioactivity was associated with A-I and A-II, respectively. In radioiodinated Lp(AI w/o AII), 71.5 +/- 6.8% (n = 6) of the total radioactivity was A-I-associated. Time-course studies showed that, while some radiolabeled proteins transferred from one population of HDL particles to another within minutes, at least several hours were necessary for transfer to approach equilibrium. Incubation of the subpopulations at equal A-I mass resulted in the transfer of 51.8 +/- 5.0% (n = 4) of total radioactivity from [125I]Lp(AI w/o AII) to Lp(AI w AII) at 37 degrees C in 24 h. The specific activity (S.A.) of A-I in the two subpopulations after incubation was nearly identical. Under similar incubation conditions, only 13.4 +/- 4.6% (n = 4) of total radioactivity was transferred from [125I]Lp(AI w AII) to Lp(AI w/o AII). The S.A. of A-I after incubation was 2-fold higher in particles with A-II than in particles without A-II. These phenomena were also observed with iodinated high-density lipoproteins (HDL) isolated by ultracentrifugation and subsequently subfractionated by immunoaffinity chromatography. However, when Lp(AI w AII) radiolabeled by in vitro exchange with free [125I]A-I was incubated with unlabeled Lp(AI w/o AII), the S.A. of A-I in particles with and without A-II differed by only 18% after incubation. These data are consistent with the following: (1) in both populations of HDL particles, some radiolabeled proteins transferred rapidly (minutes or less), while others transferred slowly (hours); (2) when Lp(AI w AII) and Lp(AI w/o AII) were directly iodinated, all labeled A-I in particles without A-II were transferable, but some labeled AI in particles with A-II were not; (3) when Lp(AI w AII) were labeled by in vitro exchange with [125I]A-I, considerably more labeled A-I were transferable. These observations suggest the presence of non-transferable A-I in Lp(AI w AII).     

Synthesis and evaluation of glycosylated octreotate analogues labeled with radioiodine and 211At via a tin precursor

Carbohydration of N-terminus and substitution of a threonine for the threoninol residue at the C-terminus of Tyr3-octreotide (TOC) has resulted in improved pharmacokinetics and tumor targeting of its radioiodinated derivatives. Yet, these peptides are very susceptible to in vivo deiodination due to the similarity of monoiodotyrosine (MIT) to thyroid hormone. The goal of this work was to develop octreotate analogues containing both a sugar moiety and a nontyrosine prosthetic group on which a radioiodine or 211At can be introduced. Solid-phase synthesis and subsequent modifications delivered an iodo standard of the target peptide N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-(3-iodobenzoyl)-Lys0-octreotate (GIBLO) and the corresponding tin precursor N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-[(3-tri-n-butylstannyl)benzoyl]-Lys0-octreotate (GTBLO). GIBLO displaced [125I]TOC from somatostatin receptor subtype 2 (SSTR2)-positive AR42J rat pancreatic tumor cell membranes with an IC50 of 0.46 +/- 0.05 nM suggesting that GIBLO retained affinity to SSTR2. GTBLO was radiohalogenated to [131I]GIBLO and N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-(3-[211At]astatobenzoyl)-Lys0-octreotate ([211At]GABLO) in 21.2 +/- 4.9% and 46.8 +/- 9.5% radiochemical yields, respectively. From a paired-label internalization assay using D341 Med medulloblastoma cells, the maximum specific internalized radioactivity from [131I]GIBLO was 1.78 +/- 0.8% of input dose compared to 9.67 +/- 0.43% for N(alpha)-(1-deoxy-D-fructosyl)-[125I]iodo-Tyr3-octreotate ([125I]I-Gluc-TOCA). Over a 4 h period, the extent of internalization of [131I]GIBLO and [211At]GABLO was similar in this cell line. In D341 Med murine subcutaneous xenografts, the uptake of [125I]I-Gluc-TOCA at 0.5, 1 and 4 h was 21.5 +/- 4.0% ID/g, 18.8 +/- 7.7% ID/g, and 0.9 +/- 0.4% ID/g, respectively. In comparison, these values for [131I]GIBLO were 6.9 +/- 1.2% ID/g, 4.7 +/- 1.4% ID/g, and 0.8 +/- 0.5% ID/g. Both in vitro and in vivo catabolism studies did not suggest the severance of the lys0 along with its appendages from the peptide. Taken together, although GIBLO maintained affinity to SSTR2, its tumor uptake both in vitro and in vivo was substantially lower than that of I-Gluc-TOCA suggesting other factors such as net charge and overall geometry of the peptide may be important.     

In vitro and in vivo binding of (E)- and (Z)-N-(iodoallyl)spiperone to dopamine D2 and serotonin 5-HT2 neuroreceptors

Apparent affinities (Ki) of (E)- and (Z)-N-(iodoallyl)spiperone [E)- and (Z)-NIASP) for dopamine D2 and serotonin 5-HT2 receptors were determined in competition binding assays. (Z)-NIASP (Ki 0.35 nM, D2; Ki 1.75 nM, 5-HT2) proved slightly more potent and selective for D2 sites in vitro than (E)-NIASP (Ki 0.72 nM, D2; Ki 1.14 nM, 5-HT2). In vivo, radioiodinated (E)- and (Z)-[125I]-NIASP showed regional distributions in mouse brain which are consonant with prolonged binding to dopamine D2 receptors accompanied by a minor serotonergic component of shorter duration. Stereoselective, dose-dependent blockade of (E)-[125I]-NIASP uptake was found for drugs binding to dopamine D2 sites, while drugs selective for serotonin 5-HT2, alpha 1-adrenergic and dopamine D1 receptors did not inhibit radioligand binding 2 hr postinjection. Specific binding in striatal tissue was essentially irreversible over the time course of the study, and (E)-[125I]-NIASP gave a striatal to cerebellar tissue radioactivity concentration of 16.9 to 1 at 6 hr postinjection. Thus, (E)-[125I]-NIASP binds with high selectivity and specificity to dopamine D2 sites in vivo.     

Androgen and estrogen dynamics in the female baboon (Papio anubis)

Androgen and estrogen dynamics were studied in 5 female baboons (Papio anubis) using constant infusions of [3H]androstenedione/[14C]estrone and [3H]testosterone/[14C]estradiol. Blood samples were obtained prior to the infusions and both blood and plasma was used for measurements of androstenedione (A), testosterone (T), dihydrotestosterone (DHT), estrone (E1), estradiol (E2). Plasma was used for measurements of sex-hormone binding globulin (SHBG), and the percents of T and E2 free, bound to SHBG, and to albumin. Blood samples obtained during the infusions were analyzed for radioactivity as purified androgens and estrogens. Metabolic clearance rates (MCR), and transfer factors ([rho]BB; fraction of steroid infused which is converted to and measured in blood as product) and blood production rates were calculated from whole blood data. All urine was collected for 96 h and an aliquot analyzed for radioactivity as the glucuronides of estrone and estradiol and the % peripheral aromatization calculated. The MCR's, calculated in whole blood, of A, E1, E2 and T were 53 +/- 6 1/day/kg, 39.3 +/- 3 1/day/kg, 29.9 +/- 5.2 1/day/kg and 10.1 +/- 2.3 1/day/kg, respectively. Each MCR was different (P less than 0.05) from the others. The PB of E1 was 15 +/- 2 micrograms/day and was not different from that of E2 (12 +/- 3 micrograms/day). The PB of A, 231 +/- 55 micrograms/day, was greater than that of T, 13 +/- 5 micrograms/day. The interconversions of both the androgens (18.9 +/- 3.4% vs 3.9 +/- 1.0%) and the estrogens (48.8 +/- 10.7% vs 4.0 +/- 0.8%) favored the oxidative pathway, i.e. conversion of 17-OH to 17-oxo steroids. The conversion ratio of A to DHT was greater than that of T to DHT (16.4 +/- 2.1% vs 5.3 +/- 0.7%), and A is a more important source of DHT than is T. The percent of T bound to SHBG (80.7 +/- 0.9%) was greater than percent of E2 (36.9 +/- 9.8%) and inversely the percents of T bound to albumin and free (17.5 +/- 0.8% and 1.65 +/- 0.16%) were less than the respective percents for estradiol (60.5 +/- 9.5% and 2.40 +/- 0.27%). The mean SHBG concentration was 54 +/- 6 nM. The peripheral aromatization of androstenedione, 1.36 +/- 0.05%, was greater than of testosterone, 0.18 +/- 0.02%. This difference is, in part, due to the lack of SHBG-binding of androstenedione. The general pattern of androgen and estrogen dynamics is similar to that in women. This similarity is due, in part, to the presence of SHBG in both baboons and women.     

Measurment of rhesus monkey (Macaca mulatta) apolipoprotein B in serum by radioimmunoassay: comparison of immunoreactivities of rhesus and human low density lipoproteins.

A sensitive and specific double antibody radio-immunoassay for the major apolipoprotein (apoB) of rhesus (Macaca mulatta) serum very low density lipoprotein (VLDL) and low density lipoprotein (LDL) is described. The anti-serum was raised to LDL (d 1.030-1.040 g/ml) and the LDL(2) (d 1.020-1.050 g/ml) was labeled with (125)I by the chloramine-T or iodine monochloride method. The assay, which was sensitive to 0.02-0.5 micro g of LDL(2), had an inter-assay coefficient of variation of 4.5%. This assay was successfully used to measure apoB in the whole serum and low density lipoproteins of control monkeys maintained on a standard Purina monkey chow (PMC) diet and of three groups of monkeys fed atherogenic diets: an "average American diet," a 25% peanut oil and 2% cholesterol-supplemented PMC diet, and a 25% coconut oil and 2% cholesterol-supplemented PMC diet. The control monkeys (n = 13) had a serum cholesterol of 146 +/- 28 mg/dl and an apoB of 50 +/- 18 mg/dl. In the monkeys maintained on the atherogenic diets the serum apoB was elevated: 103 +/- 28 mg/dl (American), 102 +/- 35 mg/dl (peanut oil), and 312 +/- 88 mg/dl (coconut oil). The values for serum total cholesterol were 333 +/- 65 mg/dl (American), 606 +/- 212 mg/dl (peanut oil), and 864 +/- 233 mg/dl (coconut oil) and were elevated relative to controls (P < 0.001). For each of the diets, total serum cholesterol correlated with serum apoB (P < 0.001). The slopes of the regression lines of serum apoB vs. cholesterol for the monkeys on the PMC, American, and coconut oil diets were similar (m = 0.531, 0.401, and 0.359, respectively), but differed from that of monkeys on the peanut oil diet (m = 0.121). The immunoreactivities of rhesus and human LDL were compared using specific antisera raised against these antigens. In homologous assay systems, monkey and human LDL exhibited unique immunological determinants. The same results were obtained with the delipidated preparations of the two LDLs using antisera raised against either monkey or human apoB. Crossover studies using a heterologous tracer with each anti-serum resulted in the selection of a specific population of antibodies directed against antigenic sites shared by these two LDL species.     

Metabolic clearance rate and half-time disappearance rate of human N-terminal and adrenocorticotropin of pro-opiomelanocortin in the rat: a comparative study

In metabolic clearance rate (MCR) and plasma half-time disappearance rate (t 1/2) of human N-terminal (1-76) and adrenocorticotropin(hACTH 1-39) of pro-opiomelanocortin were compared after intravenous bolus injection of both peptides simultaneously into rat. The level of immunoreactive (IR) hNT and IR-ACTH in plasma and urine samples were measured by specific and homologous radioimmunoassays (RIAs). The MCR and hNT and hACTH were 3.01 +/- 0.20 ml/min (M +/- S.D., N = 4) and 2.04 +/- 0.06 ml/min, respectively (p less than 0.05), The curve for the disappearance rate of IR-hNT was triphasic (rapid t 1/2 = 0.96 +/- 0.39 min, intermediate t 1/2 = 6.7 +/- 2.25 min, and slow t 1/2 = 74 +/- 15.8 min), while that of IR-ACTH was biphasic (rapid t 1/2 = 3.3 +/- 0.68 min, and slow t 1/2 = 41.5 +/- 3.03 min) as analyzed by the non-linear least-squares methods. Statistically significant difference (p less than 0.01) was found between IR-hNT and IR-hACTH in the rapid t 1/2 and in the slow t 1/2. Subsequent analysis of pooled plasma sample (30 min post-injection) by molecular sieve chromatography on Sephadex G-50 superfine column revealed that the majority of IR-hNT (90-95%) and IR-ACTH (60-70%) are co-chromatographed with [125I]iodo hNT and [125I]iodo ACTH respectively. Similarly, gel filtration of pooled urine sample (120 min post-injection) on Sephadex G-50 superfine revealed that 80-90% of IR-hNT and less than 50% of IR-ACTH co-eluted with [125I]iodo hNT and [125I]iodo ACTH, respectively. Smaller molecular forms of IR-hNT and IR-ACTH were definitely apparent in the urine sample. In conclusion, hNT has a larger MCR and a longer half-time disappearance rate (t 1/2) than IR-hACTH in rat plasma and it appears that hNT is more resistant to degradation by plasma and by kidney than hACTH.     

The control of the interaction of sex hormone-binding globulin with its receptor by steroid hormones

Sex hormone-binding globulins (SHBG) is a plasma glycoprotein that binds certain steroids. It, in turn, binds to a specific receptor on cell membranes. This work was undertaken to investigate the role of steroids in the interaction of SHBG with its receptor. Because the probe for the interaction of SHBG with its receptor is 125I-SHBG, we first showed that 125I-SHBG binds [3H]dihydrotestosterone (DHT) at 4 degrees C and 37 degrees C with KD values similar to those published previously for pure radioinert SHBG. 125I-SHBG could be prevented from binding to its receptor by a variety of steroids whose relative inhibitory activity (dihydrotestosterone much greater than 2-methoxyestradiol greater than testosterone greater than estradiol much greater than methyltrienolone greater than cortisol) was almost identical to their relative ability to bind to SHBG. Because significant binding of [3H]DHT to the SHBG receptor could not be demonstrated, steroid inhibition of SHBG binding must be noncompetitive. If steroids bound to SHBG prevent binding to the SHBG receptor, then liganded SHBG should have a higher apparent KD for its receptor than unliganded SHBG. This is the case. The KD was 0.86 +/- 0.25 nM for the high affinity receptor site using liganded SHBG and 0.19 +/- 0.024 nM for unliganded SHBG. Thus, only liganded SHBG assumes a conformation that prohibits interaction with the SHBG receptor. However, when unliganded SHBG was prebound to its receptor, it retained its ability to bind [3H] DHT. The model that emerges from these observations is as follows. Unliganded SHBG can bind either steroids or receptor in a reversible reaction; SHBG bound to a steroid cannot bind to the receptor, but unliganded SHBG that first binds to the receptor can subsequently bind steroids.     

Radioiodinated D-(+)-N1-ethyl-2-iodolysergic acid diethylamide: a ligand for in vitro and in vivo studies of serotonin receptors

Radioiodinated D-(+)-N1-ethyl-2-iodolysergic acid diethylamide ([125I]-EIL) has been evaluated as a ligand for in vitro and in vivo studies of cerebral serotonin 5-HT2 receptors. [125I]-EIL exhibited high affinity (KD = 209 pM) for 5-HT2 receptors with a high degree of specific binding (80-95%) in membranes from rat prefrontal cortex. The regional distribution of [125I]-EIL binding in vivo to seven areas of mouse brain correlated significantly (Rs = 0.93) with known densities of 5-HT2 receptors. In vivo specificity, defined by tissue to cerebellum radioactivity ratios, reached a maximum for frontal cortex at 6 hr (21.2) and persisted through 16 hr (8.8). Ketanserin, a 5-HT2 receptor antagonist, fully inhibited binding in a dose dependent fashion in all brain regions except cerebellum. By contrast, blockers for dopamine D2, alpha- or beta-adrenergic receptors did not significantly inhibit radioligand binding in any region. [125I]-EIL selectively labels 5-HT2 receptors in vivo with the highest specificity of any serotonergic ligand reported to date, indicating that [123I]-EIL should prove applicable to single photon emission computed tomography studies in living brain.     

Induction of V2 receptors in renal medulla of homozygous Brattleboro rats by arginine vasopressin

Homozygous Brattleboro rats display pronounced diabetes insipidus and when treated continuously with arginine vasopressin (AVP) acquire the ability to produce concentrated urine. In this study, the effects of continual AVP replacement on the pharmacological properties of the renal medullary V2 receptor and coupling to adenylate cyclase were examined. Osmotic minipumps that delivered AVP at four different rates were implanted into male homozygous Brattleboro rats. At the end of the 14 day treatment period, urine osmolalities were 280 +/- 24, 474 +/- 105, 1777 +/- 304 and 2202 +/- 175 mOsm/kg H2O for the 0, 31.25, 62.5 and 125 ng/hr treatment groups, respectively. Plasma AVP levels were below the level of detection for the 0 and 31.25 ng/hr treatment groups, and were 2.5 +/- 0.5 and 6.5 +/- 1.8 pg/ml for the 62.5 and 125 ng/hr treatment groups. Saturation experiments using [3H] AVP and renal medullary membranes revealed binding site concentrations of 57 +/- 9, 84 +/- 23, 164 +/- 17 and 150 +/- 18 fmol/mg protein for the 0, 31.25, 62.5 and 125 ng/hr treatment groups, respectively. AVP-stimulated cyclic AMP accumulation was enhanced in renal medullary membranes prepared from the 62.5 and 125 ng/hr treatment groups when compared to that in the 0 and 31.25 ng/hr treatment groups. From these results, it appears that circulating AVP is necessary for expression of functional V2 receptors in the homozygous Brattleboro rat renal medulla.