Insulin-like growth factor I (IGF-I) and insulin binding to erythrocytes of normal prepubertal children and adults.

Erythrocyte insulin-like growth factor I (IGF-I) and insulin receptors were characterized in 10 normal prepubertal children (5 girls and 5 boys) aged 4-11 yrs and 10 normal adults (4 women and 6 men) aged 32-47 yrs. erythrocytes were purified from 5 ml of blood by Ficoll-Paque gradient centrifugation. Reticulocytes count in the erythrocyte suspensions were lower than 1%. Insulin and IGF-I binding assays were performed simultaneously. Maximal percent binding of [125I] labelled IGF-I was significantly higher in prepubertal children than in adults (8.7 +/- 0.7% versus 6.2 +/- 0.5% at a concentration of 5 x 10(9) erythrocytes/ml). Scatchard analysis revealed the high affinity constant was better in prepubertal children (Ka = 4.6 +/- 1.3 nM-1 versus 1.8 +/- 0.2 nM-1), whereas the binding capacity was similar (5.8 +/- 1.1 versus 7.7 +/- 0.8 high affinity binding sites/cell). In both groups, unlabelled IGF-I inhibited tracer-binding half maximally at about 1 nM. Insulin was 100-fold less potent. In adults, specific binding of [125I] labelled IGF-I was higher in women (7.6 +/- 0.7%) than in men (5.3 +/- 0.4%). No significant difference was observed in maximal specific binding of [125I] labelled insulin between prepubertal children (8.2 +/- 0.5%) and adults (7.2 +/- 0.7%). In both groups, competition by unlabelled insulin for [125I] labelled insulin binding gave 50% displacement for approximately 0.25 nM and IGF-I was about 80-fold less potent. Both IGF-I and insulin binding parameters were not significantly correlated with plasma hormone levels. In prepubertal children, the high-affinity IGF-I receptors number decreased with increasing high-affinity insulin receptors number.(ABSTRACT TRUNCATED AT 250 WORDS)     

The erythrocyte insulin receptor response to insulin induced hypoglycaemia

The response of the erythrocyte insulin receptor to a prolonged intravenous infusion of insulin has been measured in normal individuals during hypoglycaemia and when hypoglycaemia was prevented by the concurrent infusion of glucose. When euglycaemia was maintained, mean (+/- S.D.) specific insulin binding following the 5 hour insulin infusion was unchanged (6.9 +/- 2.1 to 6.65 +/- 2.2% bound per 2.25 X 10(9) erythrocytes). In the presence of mild hypoglycaemia, mean (+/- SD) specific insulin binding rose from 6.6 +/- 2.3 to 7.6 +/- 2.5% bound per 2.25 X 10(9) erythrocytes (P less than 0.01), after 5 hours. This increase was due to increased receptor affinity. It was not correlated with the increase in the concentration of any individual counter-regulatory hormone. Initial insulin receptor binding correlated strongly with the subsequent decline in plasma glucose concentration (r = 0.9527; P less than 0.01). Thus, acute hyperinsulinaemia, when associated with hypoglycaemia, does not result in downregulation of insulin receptors on erythrocytes but rather results in increased receptor binding. Consequently, the insulin receptor may not play an active role in protecting the individual against acute hypoglycaemia.     

Effect of N-benzoyl-D-phenylalanine and metformin on insulin receptors in neonatal streptozotocin-induced diabetic rats: studies on insulin binding to erythrocytes

In the present study, we focused on the insulin-receptor binding in circulating erythrocytes of N-benzoyl-D-phenylalanine (NBDP) and metformin in neonatal streptozotocin (nSTZ)-induced male Wistar rats. We measured blood levels of glucose and plasma insulin and the binding of insulin to cell-membrane ER receptors in NBDP and metformin-treated diabetic rats. The mean specific binding of insulin to ER was significantly lower in diabetic control rats (DC) (53.0 +/- 3.1%) than in NBDP (62.0 +/- 3.1%), metformin (66.0 +/- 3.3%) and NBDP and metformin combination-treated (72.0 +/- 4.2%) diabetic rats, resulting in a significant decrease in plasma insulin. Scatchard plot analysis demonstrated that the decrease in insulin binding was accounted for by a lower number of insulin receptor sites per cell in DC rats when compared with NBDP and metformin-treated rats. High-affinity (Kd1), low-affinity (Kd2), and kinetic analysis revealed an increase in the average receptor affinity in ER from NBDP and metformin-treated diabetic rats having NBDP 2.0 +/- 0.10 x 10(-10) M(-1) (Kd1); 12.0 +/- 0.85 x 10(-8) M(-1) (Kd2), Metformin 2.1 +/- 0.15 x 10(-10) M(-1) (Kd1); 15.0 +/- 0.80 x 10(-8) M(-1) (Kd2), NBDP and metformin 2.7 +/- 0.10 x 10(-10) M(-1) (Kd1); 20.0 +/- 1.2 x 10(-8) M(-1) (Kd2) compared with 0.9 +/- 0.06 x 10(-10) M(-1) (Kd1); 6.0 +/- 0.30 x 10(-8) M(-1) (Kd2) in DC rats. The results suggest an acute alteration in the number of insulin receptors on ER membranes in nSTZ induced diabetic control rats. Treatment with NBDP along with metformin significantly improved specific insulin binding, with receptor number and affinity binding reaching almost normal non-diabetic levels. The data presented here show that NBDP along with metformin increase total ER membrane insulin binding sites with a concomitant significant increase in plasma insulin.     

Erythrocyte insulin binding in normal infants, children and adults

To establish normal insulin binding criteria, we studied the binding of insulin to erythrocytes from normal subjects of different ages. Insulin binding to cord erythrocytes and to erythrocytes from infants aged 2-7 days was significantly higher at tracer and physiological insulin concentrations than was binding to cells from children aged 1-15 years and adults. In infants aged 1-12 months the maximum insulin binding to erythrocytes was significantly higher than that to erythrocytes from children, and in addition, it correlated negatively with age. An increase in receptor concentration was found in cord erythrocytes whereas an increased receptor affinity for insulin was found in erythrocytes from infants. Insulin binding characteristics in erythrocytes from prepubertal and pubertal children were basically similar to those in women. Erythrocytes from men bound significantly higher amounts of insulin than did those from women. This difference was associated with changes in receptor affinity for insulin. There was no correlation between the insulin binding characteristics and the circulating concentration of insulin or C-peptide. The increased erythrocyte insulin binding at birth persisted over the neonatal period. There was an overall negative correlation between the maximum insulin binding and age in the subjects studied, but the major decrease in erythrocytes insulin binding occurred during the first year of life past the neonatal period. These observations stress the importance of using age-matched controls in studies on erythrocyte insulin binding in disease states.     

Insulin binding to circulating erythrocytes in nonobese diabetic mice

Insulin binding to circulating erythrocytes was studied in nonobese diabetic (NOD) mice which develop insulinopenic diabetes mellitus spontaneously. NOD mice with a short duration of diabetes mellitus and mild insulinopenia did not show any change in insulin binding, while those with a long duration of diabetes mellitus and severe insulinopenia showed an increase in insulin binding compared with nondiabetic NOD mice (6.85 +/- 0.38% bound vs. 4.19 +/- 0.24% bound, p less than 0.01). This increase in insulin binding was due to an increase in the number of receptors. Insulin treatment of diabetic NOD mice significantly reduced the insulin binding by 64%, which resulted from a decrease in the number of the receptors. These results indicate that insulin binding to erythrocytes in NOD mice is controlled mostly by up-and-down regulation.     

Receptor binding and biological potency of despentapeptide insulin

The receptor binding and biological potency of despentapeptide insulin (DPI) was assessed in human adipocytes, rat adipocytes and rat hepatocytes. DPI displayed a lower affinity for binding to both human adipocytes (half-maximum displacement at 0.89 +/- 0.04 and 0.20 +/- 0.02 nmol/l for DPI and insulin respectively; P less than 0.001) and rat adipocytes (half-maximum displacement at 7.12 +/- 1.06 and 1.14 +/- 0.18 nmol/l respectively, P less than 0.05). However, although DPI was less potent than unmodified insulin in stimulating glucose uptake in rat adipocytes (half-maximal stimulation at 2.0 +/- 0.67 and 0.47 +/- 0.18 nmol/l respectively; P less than 0.05), DPI was equipotent with insulin in human adipocytes (half-maximal stimulation at 0.034 +/- 0.001 and 0.027 +/- 0.001 nmol/l respectively; P greater than 0.2). In rat hepatocytes, DPI was twofold less potent in binding displacement activity (half-maximum displacement at 3.8 +/- 0.9 and 1.7 +/- 0.3 nmol/l respectively; P less than 0.01) but appeared to be equivalent in stimulating amino butyric acid uptake (half-maximum stimulation at 0.98 +/- 0.12 and 0.95 +/- 0.26 nmol/l respectively). The difference in affinity of DPI binding to rat liver membranes was less marked (1.3 fold decreased compared with insulin: 5.3 +/- 0.7 and 4.2 +/- 0.6 nmol/l respectively; P less than 0.001). Thus, the decreased receptor affinity of DPI was reflected in decreased biological potency in rat adipocytes, but not in human adipocytes nor rat hepatocytes. These data suggest differences in the binding-action linking in the cells of different tissues and different species.     

Distribution of insulin receptors in human erythrocyte membranes. Insulin binding to sealed right-side-out and inside-out human erythrocyte vesicles

Analyses of insulin binding to human erythrocytes and to resealed right-side-out and inside-out erythrocyte membrane vesicles have revealed that high affinity insulin binding receptors are present on both sides of the erythrocyte membranes. Insulin binding to human erythrocytes was examined with the use of a binding assay designed to minimize the potential errors arising from the low binding capacity of this cell type and from non-specific binding in the assay. Scatchard analysis of equilibrium binding to the cells revealed a class of high affinity sites with a dissociation constant (Kd) of (1.5 +/- 0.5) X 10(-8) M and a maximum binding capacity of 50 +/- 5 sites per cell. Interestingly, both resealed right-side-out and inside-out membrane vesicles exhibited nearly identical specific sites for insulin binding. At the high affinity binding sites, for both right-side-out and inside-out vesicles, the dissociation constant (Kd) was (1.5 +/- 0.5) X 10(-8) M, and the maximum binding capacity was 17 +/- 3 sites per cell equivalent. These findings suggest that insulin receptors are present on both sides of the plasma membrane and are consistent with the participation of the erythrocyte insulin receptors in an endocytic/recycling pathway which mediates receptor-ligand internalization/externalization.     

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.     

Effect of tetrahydrocurcumin on insulin receptor status in type 2 diabetic rats: studies on insulin binding to erythrocytes

Curcumin is the most active component of turmeric. It is believed that curcumin is a potent antioxidant and anti-inflammatory agent. Tetrahydrocurcumin (THC) is one of the major metabolites of curcumin, and exhibits many of the same physiological and pharmacological activities as curcumin and, in some systems, may exert greater antioxidant activity than curcumin. Using circulating erythrocytes as the cellular mode, the insulin-binding effect of THC and curcumin was investigated. Streptozotocin (STZ)-nicotinamide-induced male Wistar rats were used as the experimental models. THC (80 mg/kg body weight) was administered orally for 45 days. The effect of THC on blood glucose, plasma insulin and insulin binding to its receptor on the cell membrane of erythrocytes were studied. Mean specific binding of insulin was significantly lowered in diabetic rats with a decrease in plasma insulin. This was due to a significant decrease in mean insulin receptors. Erythrocytes from diabetic rats showed a decreased ability for insulin-receptor binding when compared with THC-treated diabetic rats. Scatchard analysis demonstrated that the decrease in insulin binding was accounted for by a decrease in insulin receptor sites per cell, with erythrocytes of diabetic rats having less insulin receptor sites per cell than THC-treated rats. High affinity (K d1), low affinity (K d2) and kinetic analyses revealed an increase in the average receptor affinity of erythrocytes from THC-treated rats compared with those of diabetic rats. These results suggest that acute alteration of the insulin receptor on the membranes of erythrocytes occurred in diabetic rats. Treatment with THC significantly improved specific insulin binding to the receptors, with receptor numbers and affinity binding reaching near-normal levels. Our study suggests the mechanism by which THC increases the number of total cellular insulin binding sites resulting in a significant increase in plasma insulin. The effect of THC is more prominent than that of curcumin.     

Inhibition of insulin receptor binding by dimethyl sulfoxide.

Little is known of the effects of the solvent on hormone-receptor interactions. In the present study the effect of the polar solvent dimethyl sulfoxide on the binding of insulin to its surface receptors on cultured human lymphocytes of the IM-9 line was investigated. At concentrations exceeding 0.1% (v/v), dimethyl sulfoxide produced a dose-related inhibition of 125-I-labeled insulin binding. Insulin binding was totally abolished in 20% dimethyl sulfoxide. This inhibition was immediately present and was totally reversible. Analysis of the data of binding at steady state indicated that the decrease in binding of 125I-labeled insulin was due to a reduced affinity of the insulin receptor without noticeable change in the concentration of receptor sites. Kinetic studies showed that the decreased affinity could largely be accounted for by a decreased association rate constant; effects on dissociation and negative cooperativity of the insulin receptor was affected to a much lesser extent.     

Erythrocyte insulin receptor and glucose tolerance test in children treated with prednisolone

Insulin receptors of erythrocytes and oral glucose tolerance test (O-GTT) were investigated in sixteen children treated with prednisolone for various diseases. Ten patients (Group 1) received low doses of prednisolone (0.2-0.5 mg/kg body weight/day) and six patients (Group 2) received higher doses of prednisolone (1.5-2.0 mg/kg body weight/day). Compared to the values for controls, the sums of blood glucose (sigma BS) at O-GTT in both group 1 and group 2 patients were significantly elevated. (422 +/- 75 mg/dl, p less than 0.01 Group 1; 419 +/- 39 mg/dl, p less than 0.01 Group 2; 338 +/- 41 mg/dl controls) Significant differences were not observed in the sums of insulin concentration at O-GTT, fasting blood concentration and basal insulin levels among these two groups and the controls. There was a significant increase in the maximum insulin binding in group 2 (9.13 +/- 0.68% in group 2, 7.97 +/- 1.06% in controls, p less than 0.05), but not in group 1 (8.59 +/- 1.82%). There is no significant difference in binding affinity or the number of receptors between any of these two patients' groups and the controls. When patients in group 1 and group 2 were combined, sigma IRI levels were significantly elevated in the patients (p less than 0.05). These results suggested that prednisolone treatment with a smaller dosage as well as with the higher dosage resulted in a carbohydrate intolerance, the main cause of which is located in a postreceptor step (or steps) of insulin action.     

Insulin binding and degradation in isolated hepatocytes from streptozotocin injected rats

Isolated hepatocytes from streptozotocin injected rats bound the same amount of [125I]monoiodoinsulin as hepatocytes from control rats. Scatchard analysis confirmed that insulin receptor number and affinity were the same for both groups. Relatively more cell-associated radioactivity was located intracellularly in hepatocytes from streptozotocin injected rats. Pretreatment with chloroquine resulted in a smaller increase in intracellular [125I]monoiodoinsulin in cells isolated from streptozotocin injected rats than for control cells. These results suggest that intracellular insulin processing occurs more slowly in hepatocytes isolated from streptozotocin injected rats than from control rats.     

High affinity insulin binding and insulin receptor-effector coupling: modulation by Ca2+.

Insulin binding and insulin stimulated amino acid and glucose uptake were determined in cultured HTC hepatoma cells in the presence of Ca2+ and ruthenium red (RR) in order to further characterise the putative calcium binding site on the receptor. These ions increased insulin receptor high affinity binding and the sensitivity of these responses to insulin. The insulin concentration required to half-maximally stimulate amino acid uptake decreased significantly from 26.9 +/- 5.8 ng/ml to 6.0 +/- 1.3 ng/ml in the presence of 10 mM Ca2+ and to 1.3 +/- 0.5 ng/ml in the presence of RR. The effect of Ca2+ and RR was more pronounced on insulin stimulated glucose uptake. These agents also increased receptor-effector coupling, reducing the percentage of occupied receptors required for maximal insulin stimulation of amino acid uptake from 10.8% in control cells to 3.4 and 1.4% in the presence of Ca2+ and RR respectively. The receptor occupancy required to produce maximal insulin responses on glucose uptake decreased from 20% (control) to 3.8% (Ca2+ and RR). We hypothesize that since Ca2+ and RR have similar effects, that occupation of Ca2+ binding sites on the receptor produces a conformational change in the insulin receptor which increases insulin receptor affinity, insulin sensitivity and acts on an early post-receptor event responsible for coupling binding to insulin action.     

Inhibition of insulin receptor binding by dimethyl sulfoxide

Little is known of the effects of the solvent on hormone-receptor interactions. In the present study the effect of the polar solvent dimethyl sulfoxide on the binding of insulin to its surface receptors on cultured human lymphocytes of the IM-9 line was investigated. At concentrations exceeding 0.1% (v/v), dimethyl sulfoxide produced a dose-related inhibition of ¹²⁵I-labeled insulin binding. Insulin binding was totally abolished in 20% dimethyl sulfoxide. This inhibition was immediately present and was totally reversible. Analysis of the data of binding at steady state indicated that the decrease in binding of ¹²⁵I-labeled insulin was due to a reduced affinity of the insulin receptor without noticeable change in the concentration of receptor sites. Kinetic studies showed that the decreased affinity could largely be accounted for by a decreased association rate constant; effects on dissociation and negative cooperativity of the insulin receptor were affected to a much lesser extent.     

Insulin receptor autophosphorylation and kinase activity in streptozotocin diabetic rats. Effect of a short fast

Insulin receptor associated kinase activity and its relationships with the insulin resistance of streptozotocin-induced diabetes were investigated in rats, using solubilized, partially purified insulin receptors from liver membranes. Insulin receptor kinase activity was measured by means of both autophosphorylation and phosphorylation of the exogenous substrate Glu4:Tyr1. Diabetes was associated with a 45% reduction in kinase activity, in the same number of insulin receptors, with no change in insulin binding affinity. To investigate the independent roles of hyperglycemia and hypoinsulinemia on the observed impairment of receptor kinase activity, diabetic rats were fasted for 24 h in order to normalize blood glucose levels only. After this short fast, no change in kinase activity, from the values measured in fed diabetic animals, was observed. Our findings suggest that streptozotocin diabetes is associated with a reduction of insulin receptor kinase activity, which a short fast is not able to reverse.     

Time dependent effects of dexamethasone on serum insulin level and insulin receptors in rat liver and erythrocytes

The effects of glucocorticoid excess on regulation of insulin receptors were investigated in dexamethasone-treated rats. Glucocorticoid excess was produced by administration of dexamethasone (0.5 mg/100 g b.w.) 30 min, 4, 12, 18, 24, 42 or 70 h before experiments. This treatment caused time-dependent changes of glucose and insulin concentration in blood, as well as in amounts of specific insulin binding and insulin receptors of liver cells and erythrocytes. The time intervals in which dexamethasone produced the increase in insulin concentration were accompanied with decrease in insulin binding to receptors in membranes of liver cells, while significant changes in insulin binding to receptors of erythrocytes were not observed under the same experimental conditions. The effect is maximal 18 and 42 h after dexamethasone treatment that increase insulin blood level by about 85% and 60%, respectively. Receptor analysis revealed that changes in specific binding of insulin could be due to significant changes in amount of binding sites on cell surface rather than to mild alteration in receptor affinity. These findings suggest that besides the changes in insulin level, the alterations in insulin receptor number and affinity may play a major role in the states of altered insulin sensitivity which accompany glucocorticoid excess.     

Lack of insulin effect on its own receptors in fetal rat hepatocytes

To determine the effect of insulin on its receptor concentrations in hepatocytes of fetal and adult rats, these cells were preincubated in the presence or absence of insulin. The reduced [125I]-insulin binding observed in adult hepatocytes was dependent on the concentration of insulin and on the duration of exposure, while in fetal hepatocytes insulin did not induce any reduction in insulin binding. In contrast, glucagon receptors were unaffected by preincubation with insulin. The modifications observed in insulin binding were accounted for by changes in receptor concentrations rather than any change in receptor affinity for the hormone. Studies on the kinetic properties of the insulin receptors of fetuses and adult rats revealed that association and dissociation rates were undistinguishable. These results indicate an absence of insulin receptor down-regulation in the fetus, which could favour anabolic processes during intrauterine life.     

Insulin hyperresponsiveness in partially hepatectomized diabetic rats

The present work analyzes the expression of insulin receptors and theirs related intracellular signaling molecules in partially hepatectomized-diabetic rats. Insulin binding through Scatchard analysis was studied using isolated hepatocytes of Control (Sham-operated), Hepatectomized, Diabetic and Diabetic-Hepatectomized male Wistar rats. In a set of in vivo experiments, the levels of alpha subunit of the insulin receptor, the insulin receptor substrate 1 (IRS-1) and the phosphatidylinositol 3-kinase (PI3K) were determined. [3H]-thymidine incorporation into DNA 24 or 48 h after surgery was assessed in all the experimental groups. Scatchard analysis showed that insulin receptor number was increased in diabetic and in hepatectomized rats in the same extent (64%, with respect to Controls). Diabetic-hepatectomized rats showed a dramatic increase of the receptor concentration (400%) and on the affinity constant (532%). Besides, the insulin receptor expression was increased in the treated groups, being the higher values those of the diabetic-hepatectomized rats. IRS-1 and PI3K showed similar increases. DNA synthesis was not impaired by the diabetes state. In conclusion, increased expression of IR and IRS-1 leads to increased association of PI3K in vivo in diabetic regenerating rats. The enhancement of this pathway may reveal an insulin hyperresponsiveness in these animals.     

Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [125I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density.     

Calcium mediates the interconversion between two states of the liver inositol 1,4,5-trisphosphate receptor

D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) regulates intracellular Ca2+ by mobilizing Ca2+ from a non-mitochondrial store. We have investigated the effects of Ca2+ on the binding of [32P]Ins (1,4,5)P3 to permeabilized rat hepatocytes and a liver plasma membrane-enriched fraction. Increasing the free Ca2+ concentration in the medium from 0.1 nM to 0.7 microM increased the capacity of a high affinity binding component (KD = 2-3 nM) in permeabilized cells by a factor of 10. If the membrane fraction was preincubated at 37 degrees C before binding was measured at 4 degrees C, all of the Ins(1,4,5)P3 receptors were transformed to a low affinity state (KD = 65 +/- 12 nM, Bmax = 3.1 +/- 0.1 fmol/mg, n = 4). When 0.7 microM of Ca2+ was added, the receptors were totally transformed to a high affinity state (KD = 2.8 +/- 0.4 nM, Bmax = 2.7 +/- 0.4 fmol/mg, n = 4). The EC50 of the Ca2(+)-induced interconversion of the Ins(1,4,5)P3 receptor was 140 nM. This Ca2(+)-induced transformation of the Ins(1,4,5)P3 receptor from a low affinity to a high affinity state was associated with an inhibition of the Ins(1,4,5)P3-induced Ca2+ release in permeabilized hepatocytes. These data suggest that the Ins(1,4,5)P3-dependent hormones, by increasing the intracellular Ca2+ concentration, induce a reversible transformation of the receptor from its low affinity state, coupled to the Ca2+ release, to a desensitized high affinity state. Transformation of the receptor may play a role in the oscillatory release of Ca2+ observed in single isolated hepatocytes.