Erratum

Cultured heart cells from 2–3 day old and 5–6 day old neonatal rats have been used as a model system for the characterization of carbohydrate metabolism in developing cardiac tissue. The rate of depletion of glucose from the growth medium was dependent on (1) the age of the animals from which the cultured cells were obtained, and (2) the presence and absence of serum and/or insulin in the growth medium. The glucose depletion rate in insulin and serumcontaining medium was 9.63 ± 0.96 nmol/min/mg protein for heart cell cultures from, 2 day old rats and 3.51 ± 0.68 nmol/min/mg protein in heart cell cultures from 5 day old rats. Appearance of lactate in the medium during these experiments occurred at the rates of 18.6 ± 7.9 nmol/min/mg and 6.4 ± 1.2 nmol/min/mg, respectively. In the absence of serum and insulin, the medium glucose depletion rates were 5.7 ± 1.6 and 2.2 ± 0.5 nmol/min/mg for cells derived from 2-day-old and 5-day-old rats, respectively. It is apparent from these data that immature cardiac cells depend upon glucose as a primary source of energy for muscle contraction and cellular growth, and that less-efficient energy-yielding metabolic pathways are used to obtain ATP.     

Construction of a toxic insulin molecule: Selection and partial characterization of cells resistant to its killing effects

We have constructed an insulin-diphtheria hormono-toxin which migrates as a single 29 kd band on 10% SDS polyacrylamide gel electrophoresis. This corresponds to a one to one molar ratio of the diphtheria A-chain (23 kDa) and insulin (6 kDa) molecules. The diphtheria A-chain: insulin (DTaI) hormono-toxin demonstrates cytotoxicity in V-79 Chinese hamster cells exhibiting an LD₅₀ of 1.1×10^–8M, which is 22 x more potent than whole diphtheria toxin. Also, DTaI can competitively displace [¹²⁵I]-insulin with an ED₅₀ of 1.1×10^–8 M, which is identical to the ED₅₀ of insulin (1.1×10^–8M) and showed limited cross-reactivity with the IGF-1 receptor (12% displacement of [¹²⁵I]-IGF-1 with a DTaI concentration of 1.1×10^–8 M). We have used DTaI to select conjugate-resistant clones from the V-79 Chinese hamster fibroblast parental cell line. Conjugate-resistant variants expressed insulin binding levels ranging from 8.0±2.0 fmoles/mg protein down to 3.6±0.5 fmoles/mg protein while insulin binding in the V-79 parental cell line was 11.2±0.2 fmoles/mg protein. Additionally, a number of conjugate resistant clones expressed variable ability to grow in medium containing 5% serum. The altered ability of these clones to grow in a serum-containing medium did not correlate directly with the changes observed for insulin binding. One mutant, IV-A1-j, did not grow in a serum-free defined medium containing insulin as the predominant mitogen. This IV-A1-j mutant had a lower number of insulin receptors, no change in insulin binding affinity, no change in the rate of internalization of [¹²⁵I]-insulin and no apparent difference in [¹²⁵I]-IGF-1 binding. Further, insulin-stimulated sugar transport was similar to that observed in the parental cell line. Based on these observations we suggest that 1) DTaI elicits its cytotoxicological effects through the insulin receptor trafficking pathway, 2) DTaI can be used to isolate cells altered at the level of insulin binding and/or action, and 3) signal transduction mechanisms responsible for mediating insulin-dependent cell growth can be pursued using mutants such as IV-A1-j.     

Demonstration of human platelet β-adrenergic receptors using 125I-labeled cyanopindolol and 125I-labeled hydroxybenzylpindolol

The radioiodinated pindolol analogs ¹²⁵I-labeled cyanopindolol ([¹²⁵I]CYP) and ¹²⁵I-labeled hydroxybenzylpindolol ([¹²⁵I]HBP) have been used to study binding to human platelet β-adrenergic receptors. [¹²⁵I]CYP binds to a saturable class of binding sites on platelet membranes with a dissociation constant (K_d) of 14±3 pM and maximal binding capacity (B_max) of 18±4 fmol/mg protein. Binding of [¹²⁵I]CYP is reversible and is characterized by forward and reverse rate constants of 1.8·10⁷ s^?1·M^?1 and 3.8·10^?4 s^?1, respectively. [¹²⁵I]HBP binds to a saturable class of platelet membrane sites with a K_d of 50±10 pM and B_max of 32±6 fmol/mg protein. [¹²⁵I]HBP also binds to a saturable class of sites on intact platelets with a K_d of 58±14 pM and B_max of 24±4 molecules per platelet. Binding of [¹²⁵I]CYP and [¹²⁵I]HBP is stereospecifically inhibited by propranolol and epinephrine; the (?) stereoisomers are at least 50-times more potent than the (+) stereoisomers. Binding of both radioligands is inhibited by adrenergic ligands with a potency order of propranolol ? isoproterenol > epinephrine > practolol > norepinephrine > phenylephrine. These observations indicate that [¹²⁵I]CYP and [¹²⁵I]HBP bind to platelet sites which have the pharmacological characteristics of β-adrenergic receptors but which are not typical of either the β₁ or β₂ sub-type.     

Comparison of Insulin Binding Proteins In Plasma and Nuclear Membranes of Obese and Lean Mouse Liver

Abstract

Plasma membranes obtained from obese (ob/ob) and lean (+/+ or +/ob) mouse livers were chemically crosslinked to [¹²⁵I] -insulin and examined by electrophoresis and autoradiography. The pattern of crosslinked hormone was qualitatively similar in obese and lean plasma membranes. A major insulin binding protein of approximately M 120,000 was observed. Two additional bands were apparent, one which remained near the top of the gel and one about M 90,000. A minor band at approximately M 50,000 was also detected. For each of the insulin binding proteins a reduction in the amount of [¹²⁵I]-insulin bound was observed with obese plasma membranes as compared with lean. For all proteins the insulin binding was specific as determined by competition with unlabeled hormone. In addition to plasma membrane receptors, insulin has also been reported to bind to nuclear membranes. The autoradiographic patterns of gels of [¹²⁵]-insulin bound and crosslinked to nuclear membranes from obese and lean mouse livers indicated the presence of proteins of the same M as those described for plasma membranes. Nuclear membrane proteins bound less insulin than plasma membranes and, again, the obese was decreased relative to the lean. Contamination of the nuclear membrane fraction by plasma membranes was ruled out. Scatchard analyses of [¹²⁵]-insul in bound to plasma and nuclear membranes indicated that the decrease in hormone binding in the obese mouse is a result of a reduction in the absolute number of receptors. The findings presented in this study provide additional support for this conclusion by demonstrating that membranes from obese mice are comprised of the same set of apparently unaltered insulin binding proteins. Further, the presence of similar insulin binding proteins in both nuclear and plasma membranes suggests a physiological relationship between these structures with respect to hormone binding and/or in the mechanism of action of insulin.     

Possible effect of bromocriptine on the insulin receptor

The administration of 0.00011 mg/g weight/day of bromocriptine (CB154) for 7 days to Wistar rats, improved the peripheral glucose uptake without significant changes in plasma insulin level, during the intravenous glucose tolerance test (0.33 g/kg). The mode of the bromocriptine action on binding of 125I insulin to erythrocyte insulin receptors has been evaluated. The total number of sites was greater with bromocriptine (513.1 +/- 124.1 pM/1,CB154 815.6 +/- 107.9 pM/l) (p less than 0.01). The high affinity/low capacity compound of insulin receptor, in CB154 rats (51.8 +/- 16.8 pM/l) was higher than in normal rats (18.3 +/- 8.9 pM/l) (p less than 0.005). Additional studies indicated that CB154 had no effect on the rate of association and dissociation of 125I insulin from rats erythrocyte insulin receptors. The degradation of insulin or the erythrocyte receptor sites do not change, after the treatment with CB154.     

Effect of insulin on glucose oxidation and amino isobutyric acid transport and binding of insulin in chicken thymocytes

In chicken thymocytes isolated from 15–40 day-old chickens, after a 2 h incubation at 37°C, insulin stimulated amino isobutyric acid uptake (maximal response: 40–50% of increase at 1 μg insulin/ml and half maximal response at 60 ng/ml) by specifically stimulating the influx without altering the efflux. Insulin also stimulated glucose oxidation (maximal response: 11% of increase at 1 μg insulin/ml). Binding of ¹²⁵I-labelled chicken insulin to thymocytes was rapid and higher at 15°C than at 37°C. At steady state, (90 min at 15°C), chicken, porcine and goose insulins were equipotent in inhibiting the binding of ¹²⁵I-labelled chicken insulin. Maximal binding capacity was estimated at 1250 pg insulin/10⁸ cells, i.e., 1250 binding sites/cell with an apparent dissociation constant of 200 ng insulin/ml at 15°C. Degradation of ¹²⁵I-labelled chicken insulin in the incubation medium was negligible at 15°C but very noticeable at 37°C. Therefore, the low level of insulin binding at 15°C reflects a true scarcity of insulin receptors in chicken thymocytes as compared to rat thymocytes.     

The aging leydig cell: VII. Cytoplasmic estradiol receptors

Plasma estradiol and cytosolic estradiol receptor levels of testes were determined in a group of young (2–3 months) and old (24 months) Sprague-Dawley rats. Estradiol binding sites for the young rats averaged 5.6 ± 0.3 fmol/mg protein ($\text{x}\text{±}\text{SE, n}\text{=12}$), which was comparable to that of the old rats, 5.7 ± 0.3 fmol/mg protein (n=12). Using Scatchard analyses, the association constants at equilibrium of estradiol receptor binding of the old and young rats were the same, 6.1 × 10¹⁰M^?1. Plasma estradiol levels were also similar in both groups-19.6 ± 2.8 pg/ ml (n=14) for the young and 19.2 ± 2.6 pg/ml (n=10) for the old rats. Our results suggest that impaired testosterone biosynthesis in old rats was not due to elevated plasma estradiol levels or to differences in testicular estradiol receptor content.     

Down-regulation of mt1 melatonin receptors in rat ovary following estrogen exposure.

In this study, we have demonstrated that 2-[125I]-iodomelatonin binds specifically to rat ovarian granulosa cell (GC) membranes with high affinity (KD=83 pM; Bmax=3.28 fmol/mg protein). Using immunoblot analysis and an anti-mt1 melatonin receptor antibody, we have also detected mt1 melatonin receptors in rat ovary. Because melatonin has been reported to alter the steroidogenic responses of ovarian tissues to gonadotropins, a physiological role for intra-ovarian melatonin may exist. Thus, in order to investigate a possible intra-ovarian role for melatonin, we have used both an in vivo and in vitro model of follicular development. Treatment of immature (day 21) female rats with estradiol (E; 0.2 mg/d x 3 d; subcutaneous) was used to induce follicular growth. Membranes from both untreated (U) and E-treated animals' ovaries contained high-affinity 2-[125I]-iodomelatonin (I-MEL) binding sites (Kd=83 and 23 pM, respectively). Estradiol treatment in vivo caused a significant decrease (P<0.05) in binding of 2-[125I]-iodomelatonin to ovarian membranes with untreated animals' ovaries having a Bmax=3.28 fmol/mg protein vs. estradiol-treated animals' ovaries having a Bmax=0.92 fmol/mg protein. In addition, following Estradiol treatment, mt1 melatonin receptors in rat ovary were down-regulated (approximately 95%) using immunoblot analysis. Granulosa cells isolated from E-treated rats were further matured in vitro with testosterone (T) and the pituitary gonadotropin follicle-stimulating hormone (FSH). Granulosa cells were cultured with either T (10 ng/ml) or FSH (5.71 ng ovine FSH-20/ml) alone, or both FSH and T for 48 h. There was no statistically significant specific binding of 2-[125I]-iodomelatonin to GC membranes cultured with T or FSH alone. However, following a 48-h exposure to FSH and T in vitro specific 2-[125I]-iodomelatonin binding occurred with total 2-[125I]-iodomelatonin binding =3.15 [corrected] fmol/mg protein. Therefore, the existence of hormonally-regulated expression of high-affinity melatonin binding sites suggests that melatonin may have an important intra-ovarian physiological role.     

The hepatic binding and uptake kinetics of epidermal growth factor: studies with isolated rat hepatocytes

Hepatocytes are known to bind and internalize a variety of small molecular weight proteins by a process known as receptor-mediated endocytosis (RME). The purpose of this investigation was to characterize the binding and uptake kinetics of a small protein known to be taken up by the liver by RME, epidermal growth factor (EGF), using suspensions of freshly isolated rat hepatocytes. Rat hepatocytes accumulated 125I-EGF (90 pM) in a temperature-dependent fashion. Isolated hepatocytes incubated at 37 degrees C with 125I-EGF began to release a TCA-soluble radiolabeled material into the incubation medium with a lag period of 20 min. EGF uptake by isolated hepatocytes was linear for only 60 seconds and displayed saturation kinetics (apparent Km of 4 nM and a Vmax of 105 fM/min/10(6) cells). Hepatocytes incubated at 4 degrees C bound, but did not internalize, EGF. Under these conditions, EGF binding was saturable at concentrations above 8 nM. A Scatchard analysis revealed that the average number of receptors per hepatocyte was 7.7 X 10(4) with a dissociation constant of 2.6 nM. These data demonstrate that freshly isolated hepatocytes are capable of binding, internalizing and metabolizing EGF and thus are a good model to study RME of small molecular weight proteins.     

Species differences in tears; Comparative investigation in the chimpanzee (Pan troglodytes)

This paper describes evolutionary divergence in composition of tear fluid among some mammals, and discusses the implications of these differences with regard to the choice of appropriate animals for use as models in ophthalmic research. For the first time a comprehensive investigation of tear fluid in the chimpanzee (Pan troglodytes) is presented in which tear fluid was collected during narcosis of eight chimpanzees. Total protein in chimpanzee tear fluid (8.8±0.3 g/l) is not significantly different from total protein of human tear fluid (10.0±0.6 g/l). The values in tear fluid for lysozyme (6.2±1.5 mg Hen Egg Lysozyme, HEL/ml), peroxidase (115±18 U/ml), and amylase (3.5±0.4 U/ml) in chimpanzees were significantly different from those of human lysozyme (11.8±1.6 mg HEL/ml), peroxidase (70±5 U/ml), and amylase (1.0±0.2 U/ml). Polyacrylamide gelelectrophoresis of tear fluid of the chimpanzees shows in comparison with human tear fluid an additional low-molecular protein (<14 kiloDalton).     

Decreased response with age of the cardiac catecholamine sensitive adenylate cyclase system

The cardiac β-adrenergic coupled adenylate cyclase system was examined in young and old male Wistar rats. The concentration of binding sites for (?) ³H-DHA in membranes prepared from cardiac ventricles was 21.1 ± 2.78 (SD) fmoles/mg protein in 3–4 month old rats (young rats) and 31.2 ± 2.20 fmoles/mg protein in 24 month old rats (old rats). The dissociation constant, K_D was 4.3 ± 1.8 nM and 6.7 ± 1.7 nM for young and old rats, respectively. Various compounds were used to study the characteristics of activation of adenylate cyclase in homogenates from cardiac ventricles. Basal adenylate cyclase was reduced 30% in old animals compared to young (6.1 pmoles/min/mg protein in 24 month vs. 8.6 pmoles/min/mg protein in 3–4 month). (?)Isoproterenol (10^?5M) alone stimulated adenylate cyclase greater than two-fold in young rats (10.6 pmoles/min/mg protein above basal) and this stimulation was 34% lower in old animals. GppNHp (100 μM), fluoride (10 mM), and forskolin (100 μM) activation of adenylate cyclase above basal was reduced 38, 37, and 34%, respectively, in the old animals. No significant changes between the two groups were noted in the apparent affinity of GppNHp either alone or in the presence of (?)isoproterenol nor in the affinities of catecholamine agonists for activation of cyclase. These results suggest a reduction in the amount of functional regulatory protein or possibly cyclase in 24 month old rat ventricular tissue compared to 3–4 month old tissue. However, this data does not rule out the possibility of altered molecular interactions of a full complement of regulatory protein (s) with β-adrenergic receptor and/or catalytic adenylate cyclase.     

Role of Hsp70 synthesis in the fate of the insulin-receptor complex after heat shock in cultured fetal hepatocytes

The influence of a mild heat shock on the fate of the insulin-receptor complex was studied in cultured fetal rat hepatocytes whose insulin glycogenic response is sensitive to heat [Zachayus and Plas (1995): J Cell Physiol 162:330–340]. After exposure from 15 min to 2 hr at 42.5°C, the amount of ¹²⁵I-insulin associated with cells at 37°C was progressively decreased (by 35% after 1 hr), while the release of ¹²⁵I-insulin degradation products into the medium was also inhibited (by 75%), more than expected from the decrease in insulin binding. Heat shock did not affect the insulin-induced internalization of cell surface insulin receptors but progressively suppressed the recycling at 37°C of receptors previously internalized at 42.5°C in the presence of insulin. When compared to the inhibitory effects of chloroquine on insulin degradation and insulin receptor recycling, which were immediate (within 15 min), those of heat shock developed within 1 hr of heating. The protein level of insulin receptors was not modified after heat shock and during recovery at 37°C, while that of Hsp72/73 exhibited a transitory accumulation inversely correlated with variations in insulin binding, as assayed by Western immunoblotting from whole cell extracts. Coimmunoprecipitation experiments revealed a heat shock-stimulated association of Hsp72/73 with the insulin receptor. Affinity labeling showed an interaction between ¹²⁵I-insulin and Hsp72/73 in control cells, which was inhibited by heat shock. These results suggest that increased Hsp72/73 synthesis interfered with insulin degradation and prevented the recycling of the insulin receptor and its further thermal damage via a possible chaperone-like action in fetal hepatocytes submitted to heat stress. © 1996 Wiley-Liss, Inc.     

Insulin receptors in developing rat liver: Acquisition of down regulation in the immediate postnatal period

Alterations in the high and low affinity insulin receptor concentrations in developing rat liver were investigated. The number of high affinity receptors in partially purified plasma membranes from fetal rats increased from Days 19 through 22 of gestation, with no further increase in binding during the postnatal period. Fetuses of diabetic rats had approximately three times as many high affinity insulin receptors as age-matched fetuses of normal rats; however, by 1 day after birth the receptor number decreased to the normal level. Neither the number of low affinity receptors nor the affinity of insulin binding to high or low affinity receptors changed during development or between offspring of normal and diabetic rats. These changes in the number of high affinity hepatic insulin receptors from prenatal animals did not correlate with the concentration of plasma insulin. When suckling pups were rendered diabetic the changes in the number of high affinity insulin receptors correlated with alterations in plasma insulin concentrations. The number of high affinity sites/microgram DNA in hepatocytes from Day 18 fetal rats was not altered when cells were cultured for 48 h in medium containing 0, 250, or 5000 μU/ml of added insulin. When cultured hepatocytes derived from 1-day-old and adult rats were maintained in medium with added insulin concentrations of 250 or 5000 μU/ml the number of high affinity receptors/microgram DNA decreased as compared to the number of high affinity receptors in hepatocytes cultured in medium with no added insulin. This decrease in receptor number was accompanied by an increase in the affinity of insulin binding to its high affinity receptors. The data show that (i) only the high affinity insulin receptor number increases in rat liver during the prenatal period, (ii) fetuses of diabetic rats show a greater increase in high affinity receptors than do fetuses of normal animals, and (iii) the phenomenon of down regulation for high affinity insulin receptors is not observed in fetal rat liver, but is acquired in the immediate postnatal period.     

Vanadate enhances insulin-receptor binding in gestational diabetic human placenta

Although vanadium is found abundantly in animal and plant kingdoms its biological effects are not clear. Vanadate compounds have been shown to normalize blood glucose levels in streptozotocin treated rats, enhance glucose oxidation and improve the sensitivity to insulin by enhanced receptor binding in rat adipocytes. The aim of the present study was to investigate the effect of vanadate, at high (0–8 mmol l^?1) and low (0–1·0 mmol l^?1) physiological concentrations, on [¹²⁵I]-insulin binding in the placenta of three groups of pateints, namely from normal (N) controls, gestational diabetics (GDM) and women with risk factors in their medical history for developing diabetes mellitus (RF). Vanadate at low concentrations (0·2–0·6 mmol l^?1) enhanced the maximal binding 2-fold in GDM placenta but only increased (up to 1·2-fold) the binding slightly at high cncentrations (5 mmol l^?1). However with placenta from normal or women at risk, vanadate increased the [¹²⁵I]-insulin binding up to 1·2-fold both at low and high concentrations. Thus it appears that vanadate augements insulin binding in the placenta from women with gestational diabetes mellitus.     

Insulin Binding to the Blood-Brain Barrier in the Streptozotocin Diabetic Rat

125I-Insulin binding to isolated brain microvessels from control, streptozotocin diabetic, and insulin-treated diabetic rats was measured. The binding was highest in the control (21.1 +/- 1.8%/mg capillary protein) and lowest in the diabetic (14.8 +/- 1.9%, p less than 0.01) animals. Administration of 2 U of protamine zinc insulin per day increased the maximum binding in the diabetic rats to 17.2 +/- 2.1%. Scatchard analyses of the binding showed that the major difference between the diabetic and the control animals was a decrease in the number of both high- and low-affinity sites in the diabetic animals. To test whether the failure of up-regulation in the hypoinsulinemic diabetic animal was related to an inherent defect in the endothelial cell or resulted from the diabetic milieu, cultured brain endothelial cells were tested for their capacity to up- and down-regulate their insulin receptors in vitro. In response to 100 ng/ml insulin for 12 h, these cells down-regulated their insulin receptors. When the insulin was removed, the insulin receptors returned to control levels. These studies showed that in vitro brain capillary endothelial cells have the capacity to increase their insulin receptors in response to a low-insulin environment, whereas in vivo the microvessels decrease their insulin receptors in response to diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of insulin infusion on glucose kinetics in normal and burned guinea pigs

The effects of a constant infusion of insulin (12 mu/kg·min for 90 min) on glucose turnover (determined by means of the primed-constant infusion of 6-³H-glucose) was evaluated in normal and burned (50% BSA) guinea pigs (gp). In burned, untreated gp, the mean plasma glucose level (gl) was increased from 129±8.2 to 205±13.7 mg/dl 90 min after burning, whereas gl was 140±14.5 mg/dl in the burned + insulin-infused animals at 90 min. The insulin infusion reduced gl from 120±5.6 to 69±5.8 mg/dl in unburned gp; the rate of glucose appearance (R_a) was reduced and the metabolic clearance rate (MCR) was increased. In the B+I gp, the insulin effectively minimized the increase in R_a which followed burning in the burned, untreated gp. However, insulin did not increase the MCR of the burned + insulin-infused group above that of the burned, untreated group. On the day following the burn, the insulin infusion decreased gl in the burned gp to the same extent as in the unburned animals and also increased MCR. We concluded that whereas there was a lack of peripheral responsiveness to the insulin infusion in the first 90 min after burning (during the shock phase), no such lack of responsiveness was evident on the second day.     

Insulin regulates protein metabolism in mouse blastocysts

Mouse blastocysts, in vitro, endocytosed 100 μg/ml ¹²⁵I-labelled bovine serum albumin (BSA) at a rate equivalent to 192 ± 27 μl/hr/mg embryonic protein over the first 20 min. Insulin stimulated this initial uptake by 30% (P < 0.05). After this time, accumulation of ¹²⁵I-labelled BSA began to plateau as the endocytosed ¹²⁵I-labelled BSA was catabolized and ¹²⁵I was released from the cells. Insulin caused an ≈?72% (P < 0.05) increase in the amount of uncatabolized ¹²⁵I-labelled BSA remaining in insulin-treated blastocysts after 2 hr as compared to control blastocysts. Insulin partially inhibited catabolism of endocytosed ¹²⁵I-labelled BSA during the first 2 hr following transfer to nonradioactive medium. After this time, degradation ceased in both control and insulin-treated blastocysts, leaving a small, uncatabolized protein pool remaining in the embryos; however, as a result of insulin's inhibitory effects on the initial catabolic rate, the uncatabolized protein pool was 30% (P < 0.05) larger in insulin-treated blastocysts after the 4 hr chase. Insulin inhibited endogenous protein degradation in blastocysts by 37% (P < 0.05). Combined with previous studies showing a 90% increase in endogenous protein synthesis in blastocysts following short-term stimulation with insulin (Harvey and Kaye, 1988), these results suggest that insulin acts to increase the endogenous protein-reserves in the embryo. Dose-response studies indicated an EC₅₀ of 0.5 pM for insulin's stimulation of ¹²⁵I-labelled BSA accumulation, consistent with action via its own receptor. Insulin-like growth factor-1 (IGF-1) also stimulated protein accumulation at concentrations similar to those observed with insulin, suggesting that IGF-1 may act via its own receptor rather than the insulin receptor to exert its effects on endocytosis. © 1993 Wiley-Liss, Inc.     

Low [3H]Cytochalasin B Binding in the Cerebral Cortex of Newborn Rat

The concentrations of glucose transporter in the cerebral cortex and brainstem of neonatal (4–7 days old) and adult rats were measured using [³H]cytochalasin B binding. There was significantly lower binding in neonatal cortex (1.9 ± 0.7 pmol/mg protein) compared to adult (8.9 ± 2.5 pmol/mg protein). Scatchard analysis indicates this difference is due to a lower B_max (neonate, 9.7 pmol/mg protein; adult, 18.6 ± 1.3 pmol/mg protein). Measurement of [³H]cytochalasin B binding in microvessels prepared from cortex of adult (28.1 ± 3.5 pmol/mg protein) and neonate (12.8 ± 1.9 pmol/mg protein) indicates a lower binding in the microvasculature of neonates, whereas no such difference was seen in the binding in microvessels prepared from adult and neonatal brainstem (adult, 11.8 ± 2.3 pmol/mg protein; neonate, 9.4 ± 2.7 pmol/mg protein). In both adult and neonate brain, there is an enrichment of glucose transporters in the microvasculature.     

Melatonin Binding Sites in Senegal Sole: Day/Night Changes in Density and Location in Different Regions of the Brain

We localized melatonin binding sites in different brain regions (optic tectum, telencephalon, cerebellum, hypothalamus, olfactory bulbs, and medulla oblongata) of Senegal sole, a species of aquaculture interest, and checked day/night changes in density (B_max) at mid‐light (ZT06) and mid‐dark (ZT18). Plasma melatonin was measured using a radioimmunoassay, while binding assays were performed using 2‐[¹²⁵I]iodomelatonin as a radioligand. Plasma melatonin concentrations were significantly lower at mid‐light (189.5±46 pg/ml) than mid‐dark (455.5±163 pg/ml). Values of B_max were statistically significantly higher in the optic tectum (5.6±0.6 and 12.3±1 fmol/mg prot, at mid‐light and mid‐dark, respectively) and in the cerebellum (7.7±1.1 and 10.6±1.3 fmol/mg prot, at mid‐light and mid‐dark, respectively). Significant day/night differences were only observed in these two tissues. These results show for the first time the distribution of melatonin binding sites within the brain of a flatfish species and their lack of down‐regulation.     

A rapid,sensitive, and easy-to-perform solid phase insulin radioimmunoassay

Accurate measurement of basal insulin release in perifusion, perfusion and low-density β-cell preparations has been difficult with present assays. A simple competitive, equilibrium, 15-hour insulin assay using ¹²⁵I-insulin with microtiter immobilized antubody, has been developed. This method, a Solid-phase-RadioImmunoAssay (SPRIA), is very sensitive and has a broad useful range (1 - 64 μU/ml). For a test series of 4 standard curves, interassay variation between controls of 1, 4, 16, and 64 μU/ml was ±5.2% (SEM) and intra-assay variation over the range of standards between 0.5 to 5.1% (SEM). Nonspecific binding was not significantly different from empty borosilicate culture tubes; 4.0 ± 0.4 and 3.5 ± 0.5 counts/minute (mean ± SEM; n = 54), respectively. This SPRIA can be used with existing γ-counters, while reducing the radioactive and glass waste presently produced by RIA (test-tube can be reused). The radioactive of unused test-tubes was compared againts test-tubes used for greater than 10 assays, values were 3.5 ± 0.5 and 4.4 ± 0.6 counts/minute (mean ± SEM; n = 54), respectively. Results of an oral glucose tolerance test (oGTT) performed on four male Wistar Fursth rats showed a close correlation between SPRIA and RIA insulin values (linear regression, r² = 0.990). This SPRIA measured plasma insulin levels from a human oGTT with a variation of ≤3.7% (SSEM0 between sample triplicates. Standard curves from the three commonly measured insulin isoforms (human, rat and porcine) showed a high correlation (mulitiple linear regression, r² = 0.998, n = 5 standard curves). In order to determine SPRIA's ability to measure acid extracts, insulin recovery from 2N acetic acid was compared against insulin recovery from Dullbecco's Modified Eagles medium (DME). The insulin recovery from 2N acetic acid was greater than 90% of that achieved with DME. in conclusion, an easy-to-perform assay which is deal for the rapid quantification of insulin from isolated islets of Langerhans, isolated β-cells, acetic acid extracts or plasma with greater sensitivity, and less waste than the conventional RIA has been developed.