Presence of a common structure in the two molecular species of mouse L cell interferon

Tryptic digests of the two molecular species of purified mouse L cell interferon, labeled with [¹²⁵I] and [³H] methionine, were analyzed chromatographically. The 40,000 dalton-species yielded 4 methionine-containing and 6 [¹²⁵I]-labeled fragments, whereas the 24,000 dalton-species gave rise to 4 methionine- and 7 [¹²⁵I]-labeled fragments. Of these, 3 methionine-containing and 3 [¹²⁵I]-labeled fragments were found chromatographically identical between the two species. These results suggest that the two distinct species of interferon contain a common polypeptide structure.     

The use of homologous and hetebologous 125 I-radioligands in the radioimmunoassay of progesterone

Eight homologous and heterologous¹²⁵ I-radioligand systems for the radioimmunoassay of progesterone were examined. Using an antiserum raised to 11α-hydroxyprogesterone 11-succinyl-bovine serum albumin, standard curves were set up with the homologous radioligands, 11α-hydroxyprogesterone 11-succinyl-[¹²⁵I]-iodotyramine, -[¹²⁵I]-iodohistamine and -[¹²⁵I]-iodotyrosine methyl ester. Heterologous bridge systems were represented by progesterone-11α-oxycarbonyl-[¹²⁵I]-iodotyrosine methyl ester and 11α-hydroxyprogesterone 11-phthalyl-[¹²⁵I]-iodotyrosine methyl ester, and heterologous site systems by progesterone-3-(O-carboxymethyl)oxime-[¹²⁵I]-iodotyramine, progesterone-12-(O-carboxymethyl) oxime-[¹²⁵ I]-iodotyramine, and progesterone-20-(O-carboxymethyl) oxime-[¹²⁵I]-iodohistamine. The preparation of the steroid derivatives and iodination by a two-phase method are described. The curves obtained from the homologous radioligands were relatively insensitive compared with a tritiated system, with the tyrosine methyl ester derivative providing a more sensitive assay than the corresponding tyramine or histamine analogues. The heterologous bridge systems gave more sensitive curves than the homologous tracers whilst the 3- and 12-(O-carboxymethyl) oxime derivatives of progesterone furnished curves as sensitive as the tritiated reference. Progesterone-20-(O-carboxymethyl)oxime-[¹²⁵I]-iodohistamine was not bound by the antibody.     

The secretion of parathormone and glycosylated proteins by parathyroid cells in culture

The secretion of radioactive peptides by dispersed porcine parathyroid cells incubated with [³H]- or [¹⁴C]amino acids, [³H]glucosamine and [³H]mannose was analyzed. After incubation, the culture medium contained radioactive parathormone, as expected, and two radioactive glycopeptides: SP I and SP II. SP I appears to be identical with $\text{parathyroid}\text{secretory}\text{protein}$, heretofore not recognized as a glycoprotein. SP II has not been previously identified. SP I, but not SP II or parathormone, was adsorbed by Concanavalin A possibly reflecting a high mannose content of this molecule. Raising the concentration of calcium in the medium suppressed the secretion of radioactive parathormone and SP I in a similar fashion but did not affect the secretion of SP II. Our results suggest that SP I may play a fundamental role in parathyroid synthetic or secretory processes.     

Role of gangliosides in gonadotropin and cholera enterotoxin stimulated steroidogenesis in isolated rat ovarian cells

Ovarian cells isolated from 26 day old rats responded to hCG (10 ng/ml) and cholera enterotoxin (100 ng/ml) $\text{in vitro}$ with a forty-five to fifty-fold increase in progesterone production. Both cholera enterotoxin and hCG-stimulated progesterone response was accompanied by a lag period. The duration of the lag period in the production of the progesterone depended on the concentration of gonadotropin or cholera enterotoxin, and with maximally stimulating dose it was 20–30 minutes. Addition of highly purified mixed gangliosides to the incubation medium abolished the stimulatory effect of cholera enterotoxin on progesterone response. In contrast, under identical experimental conditions, ganglioside addition produced no effect on progesterone response elicited by hCG or LH. Similarly mixed gangliosides did not prevent the specific binding of [¹²⁵I]hCG to the ovarian cells or to the membranes isolated from the ovary. In addition preincubation of [¹²⁵I]hCG with ganglioside did not alter the subsequent binding of the hormone to the ovarian cell surface receptor. These findings suggest that gangliosides are not involved in the hormone receptor interactions and subsequent receptor mediated physiological response.     

Diesterified Derivatives of 5-Iodo-2′-Deoxyuridine as Cerebral Tumor Tracers

With the aim to develop beneficial tracers for cerebral tumors, we tested two novel 5-iodo-2′-deoxyuridine (IUdR) derivatives, diesterified at the deoxyribose residue. The substances were designed to enhance the uptake into brain tumor tissue and to prolong the availability in the organism. We synthesized carrier added 5-[¹²⁵I]iodo-3′,5′-di-O-acetyl-2′-deoxyuridine (Ac₂[¹²⁵I]IUdR), 5-[¹²⁵I]iodo-3′,5′-di-O-pivaloyl-2′-deoxyuridine (Piv₂[¹²⁵I]IUdR) and their respective precursor molecules for the first time. HPLC was used for purification and to determine the specific activities. The iodonucleoside tracer were tested for their stability against human thymidine phosphorylase. DNA integration of each tracer was determined in 2 glioma cell lines (Gl261, CRL2397) and in PC12 cells in vitro. In mice, we measured the relative biodistribution and the tracer uptake in grafted brain tumors. Ac₂[¹²⁵I]IUdR, Piv₂[¹²⁵I]IUdR and [¹²⁵I]IUdR (control) were prepared with labeling yields of 31–47% and radiochemical purities of >99% (HPLC). Both diesterified iodonucleoside tracers showed a nearly 100% resistance against degradation by thymidine phosphorylase. Ac₂[¹²⁵I]IUdR and Piv₂[¹²⁵I]IUdR were specifically integrated into the DNA of all tested tumor cell lines but to a less extend than the control [¹²⁵I]IUdR. In mice, 24 h after i.p. injection, brain radioactivity uptakes were in the following order Piv₂[¹²⁵I]IUdR>Ac₂[¹²⁵I]IUdR>[¹²⁵I]IUdR. For Ac₂[¹²⁵I]IUdR we detected lower amounts of radioactivities in the thyroid and stomach, suggesting a higher stability toward deiodination. In mice bearing unilateral graft-induced brain tumors, the uptake ratios of tumor-bearing to healthy hemisphere were 51, 68 and 6 for [¹²⁵I]IUdR, Ac₂[¹²⁵I]IUdR and Piv₂[¹²⁵I]IUdR, respectively. Esterifications of both deoxyribosyl hydroxyl groups of the tumor tracer IUdR lead to advantageous properties regarding uptake into brain tumor tissue and metabolic stability.     

Steroidal and growth factor regulation of [3H]thymidine incorporation by cultured endosalpingeal cells of the bovine oviduct

Summary Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17β, progesterone, epidermal growth factor, and insulinlike growth factors I and II on [³H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24 h, DNA synthesis was quantified by pulsing cells with [³H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [³H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared to plastic, cells on a Matrigel substratum exhibited lower incorporation of [³H]thymidine and were unresponsive to hormones. Estradiol-17β increased [³H]thymidine incorporation slightly at 10⁻¹⁰ mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated [³H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17β. In the absence of estradiol, progesterone inhibited [³H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17β was present, progesterone stimulated [³H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [³H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors. These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth. Work conducted while on a sabbatical leave supported by the Deutsche Forschungsgemeinschaft.     

In vitro metabolism of a mixture of [4-C]pregnenolone and [17α-H]pregnenolone by polycystic ovary: Pathways of testosterone biosynthesis

When minced polycystic ovarian tissue was incubated with a mixture of [4-¹⁴C]-pregnenolone and [17α-³H]pregnenolone² and added cofactors for 25, 40 and 60 min, the following metabolites were isolated and characterized: progesterone, 17-hydroxyprogesterone, 17-hydroxypregnenolone, dehydroepiandrosterone, 4-androstenedione, and testosterone; unmetabolized substrates were also recovered. There were increased amounts of progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, and 4-androstenedione formed as the incubation time increased.     

A double-antibody radioimmunoassay for serum progesterone using progesterone-3-(O-carboxymethyl) oximino-[125I]-iodo-histamine as radioligand.

A reliable, convenient and economical radioimmunoassay (RIA) for serum progesterone has been established and tested. This procedure employs diethyl ether extraction followed by RIA utilizing rabbit anti-11 alpha-hydroxyprogesterone 11-hemisuccinyl-bovine serum albumin (progesterone-11 alpha-BSA) serum, progresterone-3-(O-carboxymethyl) oximino-[125I]-iodohistamine (progesterone-3-[125I]) as radioligand and goat anti-rabbit gamma globulin as second antibody. In conjunction with antiprogesterone-11 alpha-BSA serum, the overall assay specificity of the progesterone-3-[125I] RIA is similar to that of the [3H]-progesterone method using dextran-coated charcoal. The results of serum progesterone measurements during the menstrual cycle obtained by the progesterone-3-[125I] RIA appear comparable to those of [3H]-progesterone assays which employ similar anti-progesterone-11 alpha-BSA sera. The progesterone-3-[125I] double-antibody RIA, however, is more convenient and less expensive than the [3H]-progesterone RIA method.     

Substance P-Induced Reduction in the Initial Accumulation of Cytosolic myo-[3H]Inositol in Rat Parotid Acinar Cells Mediated by the NK1 Tachykinin Receptor

Abstract: Stimulation of rat parotid acinar cells by the tachykinin neurokinin (NK) 1 receptor agonist substance P (SP) resulted in a significant reduction in the initial accumulation of cytosolic myo-[³H]inositol. This effect was rapid, because a reduction of ～15% could be seen already at 30 s, with the maximal effect (～45%) being observed at 15 min. The response to SP stimulation Was temperature dependent, because at 4°C no reduction was found, jln addition, at 4°C, cytosolic myo-[³H]inositol represented only 10% of the labeled inositol accumulated at 37°C. The SP-induced reduct on in cytosolic ravo[³H]inositol accumulation was concentration dependent; the EC₅₀ obtained for SP was 5.8 ± 2.5 nM. Spantide [N Arg¹, D-Trp⁷⁹, Leu]SP), a SP antagonist, used at a concentration oif 10⁵ A/, gave a competitive shift of the dose-response curve to SP. Various tachykinins and their analogs were evaluated for their ability to reduce cytosolic mvo-[³H]inositol. [L-Pro⁹]SP and SP methyl ester, two highly selective agonists of NK1 receptors, reduced the initial accumulation of myo-H]inositol with EQo values of 2.3 and 67.0 nM, respectively. Long SP C-terminal fragments were more potent than shorter ones. SP N-terminal fragments and SP free acid were -without effect. [Pro⁷]NKB, a selective NKB analog, had no effect. The rank order of potency of mammalian tachykinins was SP > NKA > NKB. These findings and the close correlation between EC50 values and IC₅₀ values obtained in binding studies implicate the NK 1 receptor. In addition, stimulation of muscarinic receptors by carbachol alscp resulted in a reduction in level of cytosolic mjw-[³H]inositol, with this effect being reversed by atropine. Moreover, atropine was unable tjo alter the SP-induced reduction in cytosolic myo-[³H]inositol accumulation. Other neurotransmitters, such as glutamic acid, serotonin, chplecystokinin, neurotensin, bradykinin, and neuropeptide Y, were without effect on initial cytosolic myo-[³H]inositol accumulation. In conclusion, NK1 and muscarinic receptors seem to regulate the membrane transport of inositol in acinar cells of the rat parotid gland. Measurement of the initial accumulation of cytosolic myo-[³H]inositol in this tissue could profitably be adopted as a very simple, rapid, [sensitive, and specific biochemical procedure for screening the activity of potential agonists and antagonists at NK1 receptors.     

Simultaneous assay of muscarinic and β-adrenergic receptors using a double isotope technique

Muscarinic receptor and β-adrenergic receptor binding were measured simultaneously in a membrane fraction of bovine tracheal smooth muscle using [³H]-L-quinuclidinyl benzilate and [¹²⁵I]-(?)iodocyanopindolol. The binding characteristics, affinity and receptor density, obtained in the double receptor assay and in the control experiments were the same within experimental error. Moreover, there appears to be neither a significant influence of an excess of d,l-propranolol on [³H]-L-quinuclidinyl benzilate binding nor a significant influence of an excess of l-quinuclidinyl benzilate on [¹²⁵I]-(?)iodocyanopindolol binding. The method is advantageous where both receptors have to be assayed and where limited amounts of biological material, like in biopsy specimen, are available.     

[125I]MIBG uptake and release in different regions of the rat brain

Radioiodinated m-iodobenzylguanidine ([¹²⁵I]MIBG) and tritiated norepinephrine ([³H]NE]) uptake and release were compared, in different regions of the brain of the rat. The classification of the regions according to uptake was the same for both tracers: striatum > hypothalamus > hippocampus > cortex > brainstem. Tetrabenazine (TBZ), a granular monoamine uptake inhibitor reduced the uptake in the different regions. The inhibition rate was higher for [³H]NE uptake than for [¹²⁵I]MIBG. The spontaneous release was the same for [¹²⁵I]MIBG and [³H]NE and was the lowest in the striatum. The K⁺ stimulated release of [³H]NE was more complete than the release of [¹²⁵I]MIBG and was the most important in the striatum. From these results, it is inferred that MIBG enters the brain tissue via NE uptake mechanisms. It appears that MIBG is stored in the chromaffin granules, as NE, but also in the cytoplasm. A modified molecule derived from MIBG which would cross the blood-brain barrier, would then appear as a potential scintigraphic marker of monoamine uptake, storage and release.     

Endogenous low molecular weight inhibitors of cholinergic binding sites

Endogenous low molecular weight compounds which inhibit ligand binding to nicotinic acetylcholine receptors of neuronal membranes have been isolated from insect nervous tissue. Two distinct heat-stable, cationic inhibitory compounds with molecular weights of about 700-500 Da and below 500 Da have been identified. The active material was found to competitively inhibit [¹²⁵I]α-bungarotoxin and [³H]acetylcholine binding in a reversible, dose dependent manner. Comparative binding studies revealed that the active material also inhibits [¹²⁵I]α-bungarotoxin and [³H]acetylcholine binding in vertebrate brain, but not in the electric tissue of Torpedo. These results suggest that the endogenous inhibitors may function as modulators specific for neuronal acetylcholine receptors.     

Influence of short-term oestradiol treatment on plasma thyroid hormone kinetics in rainbow trout, Salmo gairdneri

The effects of 17β-oestradiol (E₂) on plasma kinetics of thyroid hormones (T₄, l-thyroxine; T₃, 3,5,3′-triiodo-l-thyronine) were studied in immature rainbow trout. E₂-3-benzoate (0.5 mg/100 g) was injected intraperitoneally on days 0 and 3, and on the morning of day 4 each trout received an intracardiac injection of either [¹²⁵I]T₄ and Na ¹³¹I or [^I25I]T₃. Groups of trout were bled and killed from 5 min to 4 days post-injection of tracer. E₂ did not alter the plasma T₄ level but depressed the T₄ plasma clearance rate, plasma-to-total tissue flux of T₄ and thyroidal T₄ secretion rate. Monodeiodination of T₄ to T₃ was also depressed, as judged from plasma [^I25I]T₃ and ^I25I ^? levels in [¹²⁵I]T₄-injected trout. E₂ had no major effect on T₃ plasma clearance rate but depressed the plasma T₃ level, plasma-to-total tissue flux of T₃ and the T₃ plasma appearance rate. E₂ had no influence on biliary transport of [^I25I]T₄ or [¹²⁵I]T₃. The above results suggest that E₂, at the dose range employed, depresses extrathyroidal T₄ to T₃ conversion, which may in turn decrease plasma T₄ clearance and thyroidal T₄ secretion.     

Two Saturable Recognition Sites for (-)[125I]Iodo-N6-(4-Hydroxyphenyl-Isopropyl)-Adenosine Binding on Purified Cardiac Sarcolemma

Abstract

Analysis of (-)[¹²⁵]iodo-N⁶-(4-hydroxyphenylisopropyl)-adenosine ([¹²⁵I]HPIA) binding to purified sarcolemmal preparations of guinea pig and bovine hearts revealed two classes of binding sites when unlabeled iodo-HPIA (100 μmol/1) was used as non-specific binding marker. In the presence of 1 mmol/1 theophylline, however, only the high affinity component was detected. Adenosine receptor agonists caused biphasic displacement of [¹²⁵I]HPIA binding, with a high affinity potency rank order typical of interaction with A₁-adenosine receptors. Biphasic competition curves were also observed with 8-phenyltheophylline and isobutylmethylxanthine, whereas the theophylline curve was monophasic up to 1 mmol/1. In brain membranes, specific binding of [¹²⁵I]HPIA as well as of [³H]PIA was further reduced when unlabeled iodo-HPIA replaces theophylline as the non-specific binding marker. These results suggest the presence of two [¹²⁵I]HPIA binding sites on cardiac sarcolemma and brain membranes, but receptor function can only be ascribed to the high affinity sites. The low affinity site probably represents an artefact, which is often observed when non-specific binding is defined with the unlabeled counterpart or a structurally related ligand of the radioligand used.     

Toxicity and mutagenicity of X-rays and [I]dUrd or [H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Synthesis and in vitro evaluation of radioiodinated indolequinones targeting NAD(P)H: Quinone oxidoreductase 1 for internal radiation therapy

NAD(P)H: quinone oxidoreductase 1 (NQO1) is an obligate two-electron reductase and is highly expressed in many human solid cancers. Because NQO1 can be induced immediately after exposure to ionizing radiation, we aimed to develop an NQO1-targeted radiolabeled agent to establish a novel internal radiation therapy that amplifies the therapeutic effects when combined with external radiation therapy. We designed three NQO1-targeted radioiodinated compounds including two ether linkage compounds ([¹²⁵I]1 and [¹²⁵I]2) and a sulfide linkage compound ([¹²⁵I]3) based on the selective binding of indolequinone analogs to the active site of NQO1 by the stacking effect. These compounds were successfully prepared using an oxidative iododestannylation reaction with high radiochemical yields and purity. In NQO1-expressing tumor cells, [¹²⁵I]1 and [¹²⁵I]2 were readily metabolized to p-[¹²⁵I]iodophenol or m-[¹²⁵I]iodophenol and [¹²⁵I]I⁻, whereas over 85% of the initial radioactivity of [¹²⁵I]3 was observed as an intact form at 1 h after incubation. The cellular uptake of [¹²⁵I]3 was significantly higher than those of [¹²⁵I]1 and [¹²⁵I]2. The uptake of [¹²⁵I]3 was specific and was dependent on the expression of NQO1. These data suggest that the novel NQO1-targeted radioiodinated compound [¹²⁵I]3 could be used as a novel internal radiation agent for the treatment of cancer.     

Heterogeneity of tachykinin receptors in the rabbit lung

The tachykinins, substance P (SP) and neurokinin A (NKA), are agonists for the NK₁ and NK₂ receptors, respectively. Tachykinins have various respiratory effects, including bronchoconstriction. This study characterizes tachykinin binding sites in the rabbit lung. We hypothesize that (2-[¹²⁵I]iodohistidyl¹)Neurokinin A ([¹²⁵I]NKA) interacts with NK₁ and NK₂ binding sites in the rabbit lung. The K_d determined from saturation isotherms was 0.69 X/÷1.14 nM (geometic mean X/÷ SEM) and the B_max was 4.15±0.22 femtomole/mg protein (arithmetic mean±SEM). Competitive inhibition studies with NKA, SP and various selective tachykinin agonists showed the rank order of potency: [β-Ala⁸]-Neurokinin A 4–10=SP ≫ NKA ≫ [Sar⁹,Met(O₂)¹¹]-Substance P. [β-Ala⁸]-Neurokinin A 4–10, a selective NK₂ agonist, and SP inhibition of [¹²⁵I]NKA binding were best described using a two-site model. Competitive inhibition studies using the selective nonpeptide NK₂ antagonist (SR 48968) and the selective nonpeptide NK₁ antagonist (CP 96,345) revealed K_i's of 5.5 nM and 8.1 nM, respectively. Our data therefore suggest that [¹²⁵I]NKA binds to both the NK₁ and NK₂ receptors in the lung. Special issue dedicated to Dr. Kinya Kuriyama.     

Scavenger receptor CD36 mediates uptake of high density lipoproteins in mice and by cultured cells

The mechanisms of HDL-mediated cholesterol transport from peripheral tissues to the liver are incompletely defined. Here the function of scavenger receptor cluster of differentiation 36 (CD36) for HDL uptake by the liver was investigated. CD36 knockout (KO) mice, which were the model, have a 37% increase (P = 0.008) of plasma HDL cholesterol compared with wild-type (WT) littermates. To explore the mechanism of this increase, HDL metabolism was investigated with HDL radiolabeled in the apolipoprotein (¹²⁵I) and cholesteryl ester (CE, [³H]) moiety. Liver uptake of [³H] and ¹²⁵I from HDL decreased in CD36 KO mice and the difference, i. e. hepatic selective CE uptake ([³H]¹²⁵I), declined (–33%, P = 0.0003) in CD36 KO compared with WT mice. Hepatic HDL holo-particle uptake (¹²⁵I) decreased (–29%, P = 0.0038) in CD36 KO mice. In vitro, uptake of ¹²⁵I-/[³H]HDL by primary liver cells from WT or CD36 KO mice revealed a diminished HDL uptake in CD36-deficient hepatocytes. Adenovirus-mediated expression of CD36 in cells induced an increase in selective CE uptake from HDL and a stimulation of holo-particle internalization. In conclusion, CD36 plays a role in HDL uptake in mice and by cultured cells. A physiologic function of CD36 in HDL metabolism in vivo is suggested.     

Metabolic pathways for androstanediol formation in immature rat testis microsomes

Metabolic routes from progesterone to androstanediol in washed rat testicular microsomes were investigated, with special emphasis on the importance of 4-ene-3-oxosteroids, as well as the effect of a minimal effective dose of human chorionic gonadotropin on these transformations. Incubation of equimolar concentrations of a mixture of [¹⁴C]progesterone and 17α-hydroxy[³H]progesterone resulted in a large preference of 17α-hydroxyprogesterone over progesterone as substrate for androstanediol formation. Incubation of [³H]progesterone together with [¹⁴C]androstenedione resulted in the inhibition of C-17,20-lyase and in a low ¹⁴C/³H ratio in androstanediol, indicating the preference of progesterone over androstenedione as substrate for androstanediol production. When a mixture of 17α-hydroxyl[³H]progesterone and [¹⁴C]androstenedione was incubated with the microsomes, a more than 8-fold preference of 17α-hydroxyprogesterone as substrate for androstanediol production was found. The minimal dose of human chorionic gonadotropin stimulated testosterone production but inhibited androstanediol formation and effected, in some instances, a change in the metabolic routes. It is concluded that androstanediol is produced preferentially through 17-hydroxylated C-21 steroids, and also, to a lesser extent, through C-19 steroids.     

A Study of the Cerebral Cortex Cholecystokinin Receptor Using Two Radiolabelled Probes: Evidence for a Common CCK 8 and CCK 4 Cholecystokinin Receptor Binding Site

Abstract: This study was directed at the issue of whether or not subpopulations of cholecystokinin (CCK) receptors exist within the CNS. This was achieved through the use of two radiolabelled probes, namely [¹²⁵I] Bolton-Hunter (BH) CCK 8 and [³H]pentagastrin (Boc-β-Ala CCK 4), in comparative studies under identical conditions. Both probes bound with high affinity to the mouse cerebral cortical CCK receptor binding site with apparent equilibrium dissociation constants (K_D) of 1.9 nM and 1.4 nM for [³H]pentagastrin and [¹²⁵I]BH CCK 8, respectively. The maximal binding capacity was 1.05 and 1.15 pmol/g weight for the tritium and iodinated probes, respectively. Hill analysis yielded Hill numbers close to unity, suggesting the absence of more than one binding site and the lack of cooperativity of CCK receptor binding. Kinetic studies revealed binding site homogeneity in that no evidence of multiphasic dissociation curves was seen. Computerised analysis of displacement binding data using LIGAND established that both radiolabelled probes bound to a single site, with the one-site model providing the best fit of the data. Similar rank orders of potency were obtained for various fragments of CCK 8 in competing for the CCK receptor, labelled with either probe. Both CCK 8 and CCK 4 bound with roughly equinanomolar affinity. These studies demonstrate that both CCK 8 and its shorter C-terminal fragment CCK 4 bind to a single class of high-affinity binding site, with as yet no evidence of CNS CCK receptor multiplicity.