The Male Obese Wistar Diabetic Fatty Rat Is a New Model of Extreme Insulin Resistance

The male obese Wistar Diabetic Fatty (WDF) rat is a genetic model of obesity and non-insulin dependent diabetes (NIDDM). The obese Zucker rat shares the same gene for obesity on a different genetic background but is not diabetic. This study evaluated the degree of insulin resistance in both obese strains by examining the binding and post binding effects of muscle insulin receptors in obese, rats exhibiting hyperinsulinemia and/or hyperglycemia. Insulin receptor binding and affinity and tyrosine kinase activity were measured in skeletal muscle from male WDF fa/fa (obese) and Fa/? (lean) and Zucker fa/fa (obese) and Fa/Fa (homozygous lean) rats. Rats were fed a high sucrose (68% of total Kcal) or Purina stock diet for 14 weeks. At 27 weeks of age, adipose depots were removed for adipose cellularity analysis and the biceps femoris muscle was removed for measurement of insulin binding and insulin-stimulated receptor kinase activity. Plasma glucose (13.9 vs. 8.4 mM) and insulin levels (14,754 vs. 7440 pmoI/L) were significantly higher in WDF obese than in Zucker obese rats. Insulin receptor number and affinity and TK activity were unaffected by diet. Insulin receptor number was significantly reduced in obese WDF rats (2.778 ± 0.617 pmol/mg protein), compared to obese Zucker rats (4.441 ± 0.913 pmol/mg potein). Both obese strains exhibited down regulation of the insulin receptor compared to their lean controls. Maximal tyrosine kinase (TK) activity was significantly reduced in obese WDF rats (505 ± 82 fmol/min/mg protein) compared to obese Zucker rats (1907 ± 610 fmol/min/mg protein). Only obese WDF rats displayed a decrease in TK activity per receptor. These observations establish the obese WDF rat as an excellent model for exploring mechanisms of extreme insulin resistance, particularly post-receptor tyrosine kinase-associated defects, in non-insulin dependent diabetes.     

The influence of hypo- and hyperthyreosis on insulin receptors and metabolism

Changes in thyroid status affect metabolism not only directly, but influence it also by alterations in insulin secretion and action. Despite several investigations, these effects are, however, poorly characterised or even controversial. The aim of the studies was to investigate the effect of hyperthyreosis (HT) and hypothyreosis (HPT) on insulin binding by rat liver membranes. Some metabolic parameters reflecting insulin and thyroid hormones action were also determined. HT and HPT were developed by daily administration for 3 weeks of thyroxine (T (4) ) and thiouracil (TU), respectively. Experimental hyperthyreosis and hypothyreosis caused deep changes in metabolism. The greatest alterations were observed in body and thyroid glands weight, blood triiodothyronine (T (3) ), T (4), glucose, and insulin levels, liver glycogen amount and number of insulin receptors. HT reflected in rats in slower rate of growth and in smaller thyroid glands weight. In comparison to controls, T (4) concentration in HT was almost doubled and it was reduced by about 30% in HPT. Also, T(3), insulin and glucose levels in HT were heightened. Simultaneously, binding of insulin to liver membranes was elevated in HT and reduced in HPT. In HT the number of high affinity insulin receptors (HAIRs) and low affinity insulin receptors (LAIRs) was increased, whereas in HPT the amount of HAIRs was diminished. HT caused a drastic reduction of glycogen concentration in liver, but no changes were observed for muscle glycogen. Considering lipid metabolism, only free fatty acids (FFA) level in blood was changed (in HPT), but no differences were observed in serum concentration of triglycerides and cholesterol. Several metabolic changes observed in HT and HPT seem to be the dire ct consequence of alterations of thyroid hormone concentrations. These disturbances, together with the direct effect of HT or HPT on insulin secretion, binding and action lead, in turn, to changes in the other metabolic parameters. As a result of these disturbances the adaptive mechanisms appear. One of them is change in the number of insulin membrane receptors taking place even against the well known "down-regulation" theory.     

VIP receptors in mesenteric and coronary arteries: a radioligand binding study

Using a biologically active radioligand, [Tyr(125I)10]VIP, we have identified and characterized receptors for vasoactive intestinal peptide (VIP) on membranes prepared from the rat superior mesenteric artery and bovine coronary arteries. Binding was specific, saturable, reversible and dependent on time and temperature. Scatchard analysis suggested the presence of a high and a low affinity binding site in each arterial system with the following binding constants: the rat mesenteric artery, KD = 0.22 +/- 0.02 and 13.6 +/- 7.8 nM (corresponding maximum number of binding sites, RO = 606 +/- 44 fmol/mg protein and 2.1 +/- 0.2 pmol/mg protein); bovine circumflex coronary artery, KD = 0.10 +/- 0.01 and 37.8 +/- 16.1 nM (corresponding RO = 369 +/- 65 fmol/mg protein and 2.0 +/- 0.7 pmol/mg protein); bovine left and right descending coronary arteries, KD = 0.12 +/- 0.03 and 21.3 +/- 6.4 nM (corresponding RO = 472 +/- 7 fmol/mg protein and 2.2 +/- 0.3 pmol/mg protein). The arterial VIP receptors did not recognize secretin, glucagon, apamin or bovine parathyroid hormone, and had reduced affinity for PHI, PHM and growth hormone releasing factors (GRF). These recognition properties were, by and large, similar to those seen in the bovine cerebral arteries although a between-species heterogeneity of recognition function could be deduced from the differences in the competitive binding of rat and bovine vascular VIP receptors with the corresponding species-specific GRFs.     

Down-regulation and recycling of high affinity cholecystokinin receptors on pancreatic acinar cells

First incubating dispersed acini from rat pancreas with monensin, a cation ionophore that can inhibit recycling of receptors, inhibited binding of 125I-cholecystokinin 8 (125I-CCK-8) measured during a second incubation by as much as 50%. A maximal effect of monensin required 90 min of first incubation. Detectable inhibition of binding of 125I-CCK-8 occurred with 300 nM monensin, and inhibition increased progressively with concentrations of monensin up to 25 microM. Pancreatic acini possess two classes of receptors that bind 125I-CCK-8. One class has a high affinity (Kd = 461 pM) and a low capacity for CCK (512 fmol/mg DNA); the other class has a low affinity (Kd = 47 nM) and a high capacity for CCK (18 pmol/mg DNA). First incubating acini with monensin caused an 84% decrease in the number of high affinity CCK receptors with no change in the number of low affinity CCK receptors or the values of Kd for either class of receptors indicating that there is recycling of high affinity CCK receptors but not low affinity CCK receptors. First incubating acini with monensin did not alter CCK-stimulated amylase secretion indicating that in contrast to previous conclusions, occupation of low affinity CCK receptors mediates CCK-stimulated enzyme secretion. Moreover, the biphasic dose-response curve for CCK-stimulated enzyme secretion from monensin-treated acini suggests that pancreatic acini also possess a third, previously unrecognized class of very low affinity CCK receptors.     

Binding of epidermal growth factor (EGF) and insulin to human liver microsomes and Golgi fractions

Microsomes and Golgi fractions were isolated from 13 human liver samples without local malignancy. Binding of insulin to microsomes (per cent per 0.5 mg protein) was 14.4 +/- 7.9% with two classes of receptors: K1 = 1.4 nM, R1 = 0.28 pmol/mg; K2 = 8.1 nM, R2 = 0.62 pmol/mg. The binding was insignificantly lower than in rats. Binding of EGF was only 3.4 +/- 1.7% with two classes of receptors: K1 = 1.4 nM, R1 = 0.06 pmol/mg; K2 = 10.8 nM, R2 = 0.22 pmol/mg; the binding was much lower than in rats (26.3 +/- 5.8%). Binding of insulin to Golgi fraction (per cent per 0.1 mg protein) was 5.5 +/- 0.4% with straight line Scatchard plot; Kd = 5.6 nM, Ro = 3.06 pmol/mg; it was only half of that found in rats. In one case of hepatoma, the binding of insulin to microsomes was normal but that of EGF very low.     

Characterization of insulin receptors in isolated epithelial cells of rat ventral prostate: effect of fasting

Insulin receptors have been characterized in rat prostatic epithelial cells by using [125I]insulin and a variety of physicochemical conditions. The binding data at equilibrium (2 h at 15 degrees C) could be interpreted in terms of two populations of insulin receptors: a class of receptors with high affinity (Kd = 2.16 nM) and low binding capacity (28.0 fmol mg-1 protein), and another class of receptors with low affinity (Kd = 0.29 microM) and high binding capacity (1.43 pmol mg-1 protein). Proinsulin exhibited a 63-fold lower affinity than insulin for binding sites whereas unrelated peptides were ineffective. The specific binding of insulin increased by about 50 per cent after 96 h of fasting; this increase could be explained by an increase of both the number of the high affinity-low capacity sites and the affinity of the low affinity-high capacity sites. These results together with previous studies on insulin action at the prostatic level strongly suggest that insulin may exert a physiological role on the prostatic epithelium.     

Hepatic uptake of asialoglycoprotein is different among mammalian species due to different receptor distribution

Isolated hepatocytes of rat, rabbit and guinea pig were found to take up and degrade 125I-labelled asialoorosomucoid at different rates with the rank order: rabbit greater than rat greater than guinea pig. Measurement of 125I-asialoorosomucoid binding at 4 degrees C to these hepatocytes revealed that all these cells had two classes of receptors with a major difference occurring in the number of high-affinity binding sites. The average binding affinity constants (K) and receptor concentration (N) calculated from a least-square analysis of the Scatchard plots were K1 = 1.15.10(9) M-1, K2 = 0.93.10(7) M-1, N1 = 0.049 pmol/mg cell protein and N2 = 0.27 pmol/mg cell protein for the rat; K2 = 3.16.10(7) M-1, N1 = 0.027 pmol/mg cell protein and N2 = 0.13 pmol/mg cell protein for the guinea pig and K1 = 0.74.10(9) M-1, K2 = 3.85.10(7) M-1, N1 = 0.205 pmol/mg cell protein and N2 = 0.37 pmol/mg cell protein for the rabbit hepatocytes, respectively. Measurement of the total number of cellular receptors after solubilization with Triton X-100 also revealed the same receptor concentration rank order of rabbit (5.8 pmol/mg cell protein) greater than rat (0.55 pmol/mg cell protein) greater than guinea pig (0.18 pmol/mg cell protein). Intravenous injection of 125I-asialoorosomucoid into anesthetized animals of matched body weight also indicated that the rate of plasma clearance and the rate of appearance of the degraded product of the tracer were different among these species with the same rank order as that observed with isolated hepatocytes. Thus there is a fundamental difference in the number of asialoglycoprotein receptors both on the cell surface and inside hepatocytes of these mammalian species.     

Beta-adrenergic ([3H] CGP-12177) receptors are elevated in slices of soleus muscle from CHE 147 dystrophic hamsters

We have utilized a muscle slice technique to compare the ontogeny of cell surface beta-adrenergic receptor binding in soleus and extensor digitorum longus (EDL) muscles of male Golden Syrian (GS) and Canadian Hybrid Farms 147 (CHF 147) dystrophic hamsters. Binding of the beta-adrenergic antagonist, [3H] CGP-12177 (CGP), to GS muscle slices was reversible, saturable, stereospecific and of high affinity. Bmax was higher in the soleus (2.57 +/- .12 fmol/mg wet wt) than in the EDL (1.6 +/- .17 fmol/mg wet wt) of adult animals while affinities were similar (0.35 +/- .06 and 0.24 +/- .04 nM respectively). No differences in binding characteristics were seen in EDL of GS compared to CHF 147 animals. In soleus slices frm GS hamsters, Bmax was highest at 16 days of age (5.72 +/- 0.26 fmol/mg), decreased between 16 and 29 days and remained constant until 300 days (2.51 +/- 0.52 fmol/mg). In dystrophic soleus slices, Bmax was also higher at 16 days than at any other age but receptor number decreased gradually, remaining higher than in GS until 90 days of age (p less than 0.05). The failure of beta-adrenergic receptor number to decrease at a normal rate may be implicated in the pathogenesis of hamster polymyopathy.     

Epidermal growth factor receptors in cultured human trophoblast cells from first- and third-trimester placentas

Epidermal growth factor (EGF) receptors were studied during the in vitro differentiation of human trophoblast cells from first- and third-trimester placentas. Cytotrophoblasts were isolated by enzymatic digestion and purified on a discontinuous Percoll gradient. As analyzed by flow cytometry, 5% of the cells are in the G2M phase in the early placenta and 0% in the term placenta. In culture, the cytotrophoblasts at both gestational ages flatten out, aggregate, and fuse together to form syncytiotrophoblasts. This in vitro morphological differentiation is associated with a threefold increase in the ability to bind specifically 125I-EGF. Trophoblastic cells from the term placenta have a significantly (p less than 0.005) higher receptor number (68.6 +/- 9.5 fmol/mg protein) for EGF after 2 days of culture than first-trimester cytotrophoblasts (35.8 +/- 2.3 fmol/mg protein). Scatchard plot analysis revealed two classes of binding sites with a similar affinity in both first-trimester and term placentas (9.5 x 10(9) M-1 for the high-affinity, 0.5 x 10(9) M-1 for the low affinity site). When 125I-EGF was affinity cross-linked to cytotrophoblasts, the receptors appeared as a specific band with a molecular weight of 180 kD in SDS-PAGE. This study demonstrates that the culture of cytotrophoblasts offer an appropriate model to study the modulation of EGF receptors.     

The binding of bombesin and somatostatin and their analogs to human colon cancers.

Specific receptors for bombesin/gastrin-releasing peptide, somatostatin, and EGF were investigated in 15 human colon cancer specimens. Eight of 15 clinical specimens (15%) of colon cancer showed the presence of somatostatin receptors. Octapeptide somatostatin analogs, RC-160 and RC-121, showed 10 times higher binding affinity for somatostatin receptors on colon cancer membranes than somatostatin. Analysis of 125I-Tyr4-bombesin binding data revealed the presence of specific binding sites in six (40%) specimens of human colon cancer. Scatchard analysis of 125I-labeled bombesin indicated a single class of receptors in three specimens with an apparent Kd value of 2.5 nM and two classes of receptors with high (Kd = 0.4 +/- 0.2 nM) and low affinity (Kd = 1.6 +/- 0.4 microM) in three other specimens. The 125I-Tyr4-bombesin binding capacities in the colon cancers for high affinity binding sites were from 6 to 228 fmol/mg protein and for low affinity binding sites 76 +/- 15 pmol/mg protein. None of the membrane preparations made from normal colonic mucosa specimens showed specific binding for 125I-Tyr4-bombesin. Five pseudononapeptide (psi 13-14) bombesin (6-14) antagonists, with different modifications at Positions 6 and 14, synthesized in our laboratory, inhibited the binding of 125I-Tyr4-bombesin in nanomolar concentrations. No correlation was found between the degree of differentiation and the presence of binding sites for somatostatin or bombesin. Specific binding of EGF was detected in 80% of colon cancer specimens. EGF binding capacity in colon cancer membranes was on average twice as high as in normal colon mucosa (50 +/- 21 vs 28 +/- 14 fmol/mg protein, respectively). Specific binding sites for somatostatin and EGF, but not bombesin, were also demonstrated in human colon cancer cell line HT-29. In HCT-116 colon cancer line only EGF receptors were found. These receptor findings and our in vivo studies on inhibition of colon cancer growth support the merit of continued evaluation of somatostatin analogs and bombesin/gastrin-releasing peptide antagonists in the management of colonic carcinoma.     

Alteration of somatostatin but not growth hormone-releasing factor pituitary binding sites in obese Zucker rats.

The present study was designed to determine whether the diminution of growth hormone (GH) secretion that occurs in obese Zucker rats is related to alterations of GH-releasing factor (GRF) or somatostatin (SRIF) pituitary binding sites. Cold saturation studies were performed in pituitary homogenates of 4-month-old lean and obese rats, using [125I-Tyr10]hGRF(1-44)NH2 as radioligand and [127I-Tyr10]hGRF-(1-44)NH2 as competitor, and in pituitary membrane preparations, using [125I-Tyr0, D-Trp8]SRIF14 as radioligand and [127I-Tyr0, D-Trp8]SRIF14 as competitor. In lean rats, analysis of the curves by the Ligand program revealed the presence of two distinct classes of GRF binding sites, the first being of high affinity (0.74 +/- 0.11 nM) and low capacity (118 +/- 31 fmol/mg protein), the second being of lower affinity (880 +/- 240 nM) and higher capacity (140 +/- 35 pmol/mg protein), and of a single class of SRIF binding sites (affinity: 0.40 +/- 0.12 nM; capacity: 24 +/- 6 fmol/mg protein). In obese rats, no difference was observed in GRF binding parameters for both classes of sites, but the concentration of somatostatin binding sites was reduced by 67% when compared to their lean littermates. These findings suggest that the SRIF pituitary receptors are down-regulated in obese Zucker rats and indicate that no alteration of GRF pituitary binding sites contribute to the blunted GH secretion observed in this model of obesity.     

Characterization of insulin binding to slices of slow and fast twitch skeletal muscles in the rabbit

Insulin binding was studied in rabbit semimembranosus proprius and psoas major muscles composed of slow-twitch oxidative (SO) and fast-twitch glycolytic (FG) fibers, respectively. For this purpose, we developed a technique using cryostat microtome muscle slices. Degradation of 125(I)-insulin during the incubation period was prevented by the addition of 1 mM bacitracin in the buffer. Specific binding to muscle slices plateaued by the 24 hrs. of incubation at 4 degrees C. It increased as a function of the amount of muscle, with a maximum binding occurring at about 5 mg of muscle slices. Triton X-100 has been shown to increase specific binding from a critical concentration of 10(-4) M with a maximum effect occurring at 3.3 10(-4) M. Under this condition, the binding was specific since displacement studies showed no inhibition of 125(I)-insulin binding by GH, HCG, ACTH and glucagon, whereas half maximal inhibition was achieved using 5 10(-10) M insulin, 3 10(-9) M IGF1 and 2 10(-8) M proinsulin. The analysis of the binding data yielded curvilinear Scatchard plots. The number of high affinity insulin receptors was higher in the SO muscle than in the FG muscle (4.3 +/- 0.7 vs 0.7 +/- 0.2 fmol/mg fresh muscle; P less than 0.001) with similar high affinity dissociation constants (Kd = 1.5 10(-10) M). Analogous results were obtained using muscle microsomal fractions. The differences in insulin binding might be related to the more intense metabolism of SO fibres which contract more often than FG fibres in vivo.     

Involvement of the peripheral cholinergic muscarinic system in the compensatory ovarian hypertrophy in the rat

In the present experiments, unilateral ovariectomy (ULO) induced compensatory hypertrophy (COH) of the remaining rat ovary (60%-85% increase in ovarian weight, total proteins, and total RNA and DNA). An increased thymidine uptake preceded the organ enlargement. COH was inhibited by i.p.-administered muscarinic antagonist propantheline (dose-dependently) or botulinum toxin delivered locally to the ovary. The effects were reversed by bethanecol i.p. (a muscarinic agonist). In sham ULO animals, [3H]-scopolamine binding to ovarian membranes indicated the existence of muscarinic receptors (Kd 2.5 nM, Bmax 12 fmol/mg proteins, Hill 1.0). The ovarian 1,2-diacylglycerol (DAG) was 120-150 pmol/mg tissue and did not react to carbachol in vitro (50 microM). At 15 minutes after ULO, the [3H]-scopolamine binding was unchanged (Kd 2.6 nM, Bmax 12.6 fmol/mg tissue, Hill 1.0), but the ovarian DAG was increased (280-350 pmol/mg tissue) and increased further in response to carbachol (460-550 pmol/mg tissue). After ULO, ovarian DAG remained continuously responsive to carbachol. The ULO-induced DAG increase and enhanced susceptibility to carbachol were inhibited by the botulinum toxin or atropine pretreatments. Abdominal vagotomy done immediately before ULO also inhibited the ULO-induced DAG increase and DAG responsiveness to carbachol. However, when the vagotomy was performed 10 mins after ULO, the ovarian DAG remained responsive to carbachol in vitro. The data suggest that the peripheral cholinergic system, including the ovarian muscarinic receptors, stimulates COH. This is apparently associated with the ULO-induced coupling of the ovarian muscarinic receptors to phosphoinositide (PI) breakdown. Vagus plays a role in the occurrence of the changed muscarinic receptor-PI breakdown relationship in the remaining ovary.     

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.     

Properties of vasoactive intestinal peptide-receptor interaction in rat liver membranes

The properties of the specific receptors for vasoactive intestinal peptide (VIP) in rat liver plasma membranes have been studied by using 125I-VIP as a tracer. The binding of the peptide was a reversible, saturable and specific process, as well as time and temperature dependent. Peptide inactivation was also dependent on time and temperature and remained relatively low in the standard conditions used, as it happened in the inactivation of the binding sites. The binding data were compatible with the existence of two classes of VIP receptors: a high affinity (Kd = 4.2 x 10(-10) M) and low binding capacity (1.5 pmol VIP/mg protein) class and another one of low affinity (Kd = 1.7 x 10(-7) M) and high binding capacity (38.6 pmol VIP/mg protein). The specificity of the binding sites of VIP was established from the fact that binding of 125I-VIP was inhibited by native VIP and by 60-fold higher concentrations of secretin but not by the parent hormone glucagon, by insulin or somatostatin at concentrations as high as 10(-6) M.     

Comparison of muscarinic and beta adrenergic receptors between bovine peripheral lung and tracheal smooth muscles: a striking difference in the receptor concentration

This study was undertaken to compare the activity of muscarinic and beta adrenergic receptors in bovine peripheral lung to the corresponding receptor activity in tracheal smooth muscle. We used [3H] quinuclidinyl benzilate (QNB) and [3H]dihydroalprenolol (DHA) to measure muscarinic and beta receptor activity, respectively. Binding to QNB and DHA at 25 degrees C was rapid, reversible, saturable and of high affinity. The order of potency for cholinergic and adrenergic agents competing for binding was compatible with muscarinic and beta 2 adrenergic potencies. We found that the concentration of muscarinic receptor binding sites was 37-fold greater in the tracheal muscle preparation (2805 +/- 309 fmol/mg protein) than in the peripheral lung preparation (76 +/- 28 fmol/mg protein). Unlike muscarinic receptors, the lung contained 8-fold higher concentration of the beta adrenergic receptors than did the tracheal muscle (1588 +/- 417 vs. 199 +/- 42 fmol/mg protein). The dissociation constant or the agonist's inhibitory constant (Ki) for either receptor binding site, however, was not significantly different between the two tissues. Furthermore, in vitro contraction studies showed that the response of tracheal muscle strips to methacholine was markedly greater than the response of peripheral lung strips, a finding consistent with the QNB binding result. The muscle but not the peripheral lung strip exhibited a relaxing response to epinephrine. Our data indicate a striking quantitative difference in muscarinic and beta adrenergic receptors between lung tissue and tracheal muscle, and that each receptor in the lung is qualitatively similar to the corresponding receptor in the muscle.     

Estrogen and androgen receptors in the liver of cynomolgus monkeys (Macaca fascicularis)

Estrogen receptors (ER) and androgen receptors (AR) were evaluated in the hepatic cytosol from cynomolgus macaques to determine if there were differences associated with gender and endogenous hormone secretion. Saturable, high affinity binding (Kd = 0.2-0.8 nM) was demonstrated for both ER and AR from either male or female monkeys. Displacement of tritiated estradiol from the ER was estrogen specific (including ethinyl estradiol). Both androgens and the synthetic progestins (levonorgestrel and norethindrone) displaced tritiated mibolerone from the AR. Both 8S and 4S molecular forms of ER and AR were demonstrated on 5-20% sucrose density gradients. The ER levels were higher in females in the follicular phase of the menstrual cycle (40.5 +/- 1.9 fmol/mg protein) than levels in males (26.4 +/- 4.8 fmol/mg protein; P less than 0.01) or levels in luteal phase females (31.8 +/- 2.4 fmol/mg protein; P less than 0.05). AR levels were not different between females during different phases of the menstrual cycle (65.8 +/- 4.6 and 69.5 +/- 4.3 fmol/mg protein, follicular and luteal, respectively), but there was a tendency (P less than 0.10) for the levels in males (54.4 +/- 6.6 fmol/mg protein) to be lower than female levels. The demonstration of saturable, high affinity binding of androgens and estrogens in liver tissue of these primates, along with differences associated with gender and the stage of the menstrual cycle, suggests that hepatic receptors are functional and may play an important role in hepatic protein secretion.     

Neurotensin receptors on the rat liver plasma membranes

Neurotensin (NT) is now classified as a brain-gut peptide in the central nervous system and gastrointestinal tract. In the present study, we characterized the NT receptors on the rat liver plasma membranes. The specific binding of [3H]NT was time dependent, reversible, and saturable. Scatchard analysis of the specific binding data yielded two classes of binding sites, a high affinity site and a low affinity site. The average maximum number of binding sites (Bmax) amounted to 13.3 +/- 1.1 fmol/mg protein at high affinity site and 122.3 +/- 21.5 fmol/mg protein at low affinity site, respectively. The dissociation constant (Kd) had values of 0.39 +/- 0.01 nM at high affinity site and 8.1 +/- 1.1 nM at low affinity site, respectively. The amount of specifically bound [3H]NT was significantly reduced in the presence of mono and divalent cations, EDTA, EGTA and a peptidase inhibitor bacitracin, NT1-13 competed with [3H]NT for its binding site with an IC50 of 0.19 nM at high affinity site (0.2 nM concentration of [3H]NT) and 0.7 nM at low affinity site (4.0 nM concentration of [3H]NT). Xenopsin, a NT analogue separated from the skin of Xenopus laevis, was equipotent (IC50 0.75 nM) with NT1-13 at 4.0 nM concentration of [3H]NT. C-terminal sequence of NT contains the structure necessary for interaction with NT binding sites whereas N-terminal sequence had no binding activity. Since NT has a hyperglysemic and a hypercholesterolemic effects in rats, these NT receptors on the rat liver plasma membranes may be involved in the hyperglycemia and/or hypercholesteroremia induced by NT.     

Subtypes of guanine-nucleotide-binding regulatory proteins at the locus coeruleus involved in fentanyl-induced muscular rigidity in the rat

Previous results from our laboratory have established that the G_o subtype of guanine nucleotide (GTP)-binding regulatory proteins at the locus coeruleus (LC) may participate in the elicitation of muscular rigidity by fentanyl. The present study further examined the involvement of other subtypes of GTP-binding regulatory proteins at the LC in this process, using Sprague-Dawley rats anesthetized with ketamine (120 mg/kg, i.p., with 30 mg/kg/h i.v. infusion supplements) and under mechanical ventilation. Intravenous administration of fentanyl (100 µg/kg) induced a significant increase in electromyographic signals recorded from the sacrococcygeus dorsi lateralis muscle. Power spectral analysis revealed that this was accomplished by a decrease in the mean power frequency and an increase in the root mean square values of the signals. The above responses were appreciably antagonized by pretreating animals with bilateral microinjection into the LC of pertussis toxin (80 or 160 fmol), N-ethylmaleimide (16 pmol) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (100 or 200 fmol); but not by cholera toxin (120 or 240 fmol), forskolin (240 or 480 pmol) or N-ethylmaleimide at a higher dose (32 pmol). These results suggest that, in addition to G_o protein, fentanyl-induced muscular rigidity may also involve other pertussis toxin-sensitive GTP-binding regulatory proteins, possibly G_i and G_p subtypes, in the signal transduction processes following activation of -opioid receptors at the LC.     

The receptor for epidermal growth factor is present in human fetal kidney, liver and lung

In animals a pharmacological doses of the growth-promoting peptide epidermal growth factor (EGF) has an effect on the growth and/or maturation of several organs such as the lung, the kidney, the liver and the gastrointestinal tract. Since EGF elicits its function via binding to specific cellular receptors the presence of these receptors predicts a possible physiological role for EGF and EGF agonists. We have studied the presence of the EGF-receptor on human fetal membrane preparations from the kidney, the liver, the lung and the placenta (gestational age 13-20 weeks). The 4 membrane preparations all bind labeled EGF thus allowing us to calculate the apparent affinity constant and the number of receptors present per mg of membrane protein. The apparent affinity constant (gestational age 13-20 weeks) varies between 0.5 and 3.5 X 10(9) mol-1, median 1.3 X 10(9) mol-1 (n = 40). No difference is observed for the 4 tissues examined, and no difference is found as a function of the gestational age. The number of receptors present per mg of membrane protein (gestational age 16-20 weeks) are (range and (median) 90-220 (130) fmol, n = 10 for the kidney, 80-480 (250) fmol, n = 9 for the liver, 90-690 (300) fmol, n = 10 for the lung, and 2100-4200 (3400) fmol, n = 7 for the placenta. Results for a fetus of gestational age 13 weeks show high values for kidney receptors (240 fmol) and lung receptors (800 fmol) and low values for the placenta receptors (410 fmol).