Interaction of insulin-like growth factors (IGF) with isolated sheep hepatocytes. II. Binding, internalization and degradation of IGF-II

This study examines the binding and degradation of IGF-II by the ovine liver. Binding and degradation of 125I-IGF-II to isolated hepatocytes was time, temperature and cell number dependent. Ovine and human IGF-II were 2-5 times more effective in inhibiting 125I-hIGF-II binding than were the IGF-I preparations. Insulin did not affect binding. Autoradiographs of 125I-hIGF-II affinity cross-linked to hepatocytes showed a major band of molecular weight 271,000 under reduced conditions. This band was eliminated by 100 nM hIGF-II or oIGF-II but not by excess hIGF-I, oIGF-I or insulin. The internalization of IGF-II was examined by treating the cells with trypsin or sodium acetate to remove surface-bound IGF-II. Both treatments showed that 20-25% of 125I-hIGF-II was internalized. Mannose-6-phosphate at 1, 2 and 4 mM enhanced the binding of 125I-hIGF-II to hepatocytes 3.5, 12.8 and 16.4%, respectively. The lysosomal inhibitors ammonium chloride, chloroquine and leupeptin had no effect on 125I-hIGF-II degradation or cell-associated radioactivity indicating a nonlysosomal pathway of degradation for 125I-hIGF-II in the ovine hepatocyte. The low molecular weight sheep serum IGF binding protein inhibited binding of 125I-hIGF-II in a dose-dependent manner but had no effect on degradation, which suggests that degradation of 125I-hIGF-II is independent of receptor interaction. These studies demonstrate that IGF-II binds to specific high affinity sites in sheep hepatocytes which display the characteristics of type II IGF receptors. A significant fraction of the receptor bound IGF-II is internalized but not degraded by these cells, which suggests that the biological actions of IGF-II may be exerted by an intracellular pathway in sheep hepatocytes.     

Characterization of type II insulin-like growth factor (IGF) receptors in sheep liver plasma membranes

Interactions of insulin-like growth factors (IGFs) from recombinant human and natural ovine sources with sheep liver plasma membranes have been studied. Total specific binding of 125I-hIGF-II (40%) to liver plasma membranes greatly exceeded that of 125I-hIGF-I (1.5%) after incubation at 20 C for 90 min. Binding of 125I-hIGF-II to the plasma membranes was dependent upon time, temperature and membrane concentration of the incubation. Binding of 125I-hIGF-II was only partially reversed by addition of 100 nM IGF-II (18%) or by dilution with excess buffer (36%). Competitive inhibition studies of 125I-hIGF-II binding demonstrated that IGF-II from ovine or recombinant human sources was more effective at inhibiting binding than ovine or human IGF-I. Insulin did not affect binding of 125I-hIGF-II. Plasma membranes were affinity cross-linked to 125I-IGF-II followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Following autoradiography, radioactive bands were localized at 274,000 Mr and 210,000-215,000 Mr in the presence and absence of reducing agent, respectively. This pattern was unaffected by 100 nM human or ovine IGF-I or 1,000 nM insulin, but coincubation with 100 nM human or ovine IGF-II eliminated the radioactive band. These data indicate that an IGF-II specific receptor is present in sheep liver plasma membranes which has characteristics similar to those of nonruminant Type II receptors.     

Insulin-like growth factor I receptor analysis of satellite cell-derived myotube membranes established from two lines of Targhee rams selected for growth rate

Ovine-derived fibroblasts were used to validate an insulin-like growth factor I (IGF-I) membrane-receptor binding assay system. Competitive binding using fibroblasts revealed that half-maximal inhibition of 125I-IGF-I binding by IGF-I was 2.3 nM. SDS-polyacrylamide gel electrophoresis analysis of specific protein-associated 125I-IGF-I was consistent with the migration of 125I-IGF-I-labeled Type I IGF receptor alpha-subunits at Mr 133,000 daltons. Further, the efficiency of two cell solubilization methods was examined and time-dependent binding equilibrium was determined for the membrane assay system. Satellite cell-derived myotubes were subsequently isolated from primary satellite cell cultures established from the semimembranosus muscles of high and low efficiency-of-gain (EOG) Targhee rams, and IGF-I receptor dynamics were measured. A membrane competitive binding study revealed that half-maximal inhibition of 125I-IGF-I binding was achieved by 1-ng IGF-I for low, and 10-ng IGF-I for high, EOG myotube membrane preparations. Kd values were similar between the high EOG (4.78 nM) and low EOG (2.95 nM) groups; however, receptor concentrations (Bmax) appeared to differ between groups. High EOG membrane receptor Bmax was 3.88 pmole/micrograms protein (19.87 pmole/micrograms DNA), whereas low EOG membrane receptor Bmax was 1.22 pmole/micrograms protein (9.28 pmole/micrograms DNA). These preliminary findings support the hypothesis that genetic selection for EOG results in altered satellite cell responsiveness to IGF-I.     

Comparison of insulin-like growth factor I serum binding proteins in sheep and cattle: species differences in size and endogenous binding capacities

Binding proteins (BP) for insulin-like growth factor I (IGF-I) were characterized in sheep and beef cattle serum for molecular weight (Mr) and binding characteristics. Serum was incubated with [125I] IGF-I at 37 degrees C before chromatography over a 1.6-cm X 94.0-cm column of Sephacryl S-300 (pH 7.4, 4 degrees C). Beef serum exhibited a 145 k Mr (mol. wt X 1,000) and a 35 to 39 k Mr BP. Sheep serum possessed a 170 to 190 k and a 35 to 38 k Mr protein. Binding of [125I] IGF-I was inhibited in the presence of excess unlabeled ovine somatomedin, demonstrating specific binding for each BP of both species. The high Mr component was pituitary-dependent in sheep, as evidenced by binding patterns from serum of hypophysectomized sheep. Direct binding studies of the Sephacryl-separated BP demonstrated that the native BP of high molecular weight of both species bound only minor amounts of [125I] IGF-I in a manner unrelated to BP concentration. The BP of low molecular weight of beef cows displayed a bell-shaped dose-response binding curve with maximum binding at 250 micrograms/ml BP, whereas binding to sheep BP of low molecular weight was independent of BP concentration. After chromatography on Sephadex G50 at pH 2.8, both BP from both species exhibited concentration-dependent binding that plateaued at 250 to 500 micrograms/ml of BP of low molecular weight but was curvilinear for the BP of high molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma IGF-I binding proteins in sheep: effect of recombinant growth hormone treatment and nutritional status.

In humans the IGF binding proteins (BP) are closely related to metabolic status. In this paper we have examined the influence of controlled feed intake and GH treatment on IGF binding proteins in growing lambs. Analyses were performed on plasma samples from animals maintained on two levels of feed intake (1.75% body weight as lucerne pellets or 3% body weight which is approximately equivalent to an ad libitum intake) either with or without recombinant bovine growth hormone (BST; 0.25 mg/kg body weight/day) administration. Samples used for the analyses reported in this paper were collected at 9.00 hr following 41 d of treatment. Total plasma IGF-I was increased on the higher plane of nutrition (P less than .01) and by BST (P less than .001) but only on high feed intake. IGF is associated with BP of 150 kDa and 40-50 kDa in sheep plasma. 150 kDa bound IGF-I was increased on the higher plane of nutrition (P less than .05) and by BST treatment (P less than .001) but only on the higher feed intake. By contrast no change in 40-50 kDa bound IGF-I was observed with treatment. Unbound IGF-I was also found in sheep plasma (2-5% of total) but demonstrated only minor changes in relation to treatment. Saturation analysis gave estimates of total binding capacity and saturation of the IGF-BP. In ovine plasma the binding capacity of the 150 kDa species is in excess of bound IGF (P less than .001). Saturation did not change with treatment despite the observed differences in 150 kDa bound IGF-I. Thus BP(s) contained in the 150 kDa fraction were responsive to treatment. By contrast large differences in saturation of the 40-50 kDa species were observed (P less than .001) despite little treatment dependent change in bound IGF-I. IGF-BP(s) in the 40-50 kDa fraction were elevated in the low nutrition group and suppressed on the higher feed intake resulting in near saturation. These data strongly suggest that the IGF BP are modulated according to metabolic status in the sheep.     

Interaction of insulin-like growth factor I with turkey satellite cells and satellite cell-derived myotubes

Satellite cells, isolated from the superficial pectoralis muscle of growing Nicholas tom turkeys, were cloned to obtain a pure population of myogenic cells. These cells proliferated rapidly and differentiated (fused) into myotubes typically containing 92-98% fused nuclei. Competitive binding assays were performed on near-confluent satellite cell or myotube cultures in 35 mm diameter wells by adding [125I]IGF-I along with increasing concentrations of unlabeled IGF-I, IGF-II, or insulin. Following incubation, the cultures were washed to remove the unbound hormones, solubilized with 0.5 N NaOH, and the radioactivity specifically bound was determined. Total and fused nuclei number as well as total protein were determined in parallel cultures. Our results indicate that turkey satellite cell and myotube cultures possess specific binding sites for IGF-I. Displacement of [125I]IGF-I was in the order of IGF-I greater than IGF-II greater than or equal to insulin. Although the [125I]IGF-I association constants were similar for turkey satellite cells and myotubes, a 2.8-fold decrease in the number of receptors per nuclei was observed as satellite cells differentiated into myotubes. The 50% inhibition constants for IGF-I, IGF-II, and insulin were 3.7 X 10(-9) M, 7.5 X 10(-8) M, and 8.7 X 10(-8) M for satellite cells and 3.1 X 10(-9) M, 7.5 X 10(-8) M, and 9.6 X 10(-8) M for myotubes, respectively. Receptor cross-linking analysis using disuccinimidyl suberate was performed on near-confluent satellite cell cultures incubated with [125I]IGF-I in the presence or absence of 1 X 10(-7) M IGF-I, IGF-II, or insulin. Receptor subunit species of Mr 130 kDa and 98 kDa were observed under reducing conditions (100 mM dithiothreitol) and at a Mr greater than 300 kDa (native receptor tetramer) under non-reduced conditions. Autoradiographic bands were displaced with IGF-I but not with equimolar levels of IGF-II or insulin. The results suggest that turkey satellite cells possess a type I IGF receptor.     

Increased degradation of insulin-like growth factor-I in serum from feed-deprived steers

Severe feed restriction decreases serum insulin-like growth factor I (IGF-I) concentration in animals, and this decrease is thought to be due to reduced IGF-I production in the liver. The objective of this study was to determine whether feed deprivation also increases degradation of serum IGF-I and serum levels of IGF binding protein 3 (IGFBP-3) and acid-labile subunit (ALS), which inhibit IGF-I degradation and increase IGF-I retention in the blood by forming a ternary complex with IGF-I, in cattle. Five steers had free access to pasture, and another five were deprived of feed for 60 h. Serum concentration of IGF-I and liver abundance of IGF-I mRNA at the end of the 60-h period were 50% and 80% lower, respectively, in feed-deprived steers than in fed steers. Less ¹²⁵I-labeled IGF-I remained intact after a 45-h incubation in sera of feed-deprived steers than in sera of fed steers, suggesting that serum IGF-I is more quickly degraded in feed-deprived animals. Serum levels of IGFBP-3 and ALS were decreased by 40% and 30%, respectively, in feed-deprived steers compared with fed steers. These decreases were associated with more than 50% reductions in IGFBP-3 and ALS mRNA in the liver, the major source of serum IGFBP-3 and ALS. Taken together, these results suggest that feed deprivation reduces serum concentration of IGF-I in cattle not only by decreasing IGF-I gene expression in the liver, but also by increasing IGF-I degradation and reducing IGF-I retention in the blood through decreasing IGFBP-3 and ALS production in the liver.     

Expression of insulin-like growth factor binding protein (IGFBP)-3, and the effects of IGFBP-2 and -3 in the bovine corpus luteum.

The present study was conducted to gain insight into the insulin-like growth factor (IGF) system in the bovine corpus luteum (CL). Specific aims were to measure the levels of IGF binding protein-3 (IGFBP-3) and RNA encoding IGFBP-3 in the CL throughout diestrus, and to investigate the effects of IGFBP-2 and -3 on IGF-I-stimulated progesterone (P4) production and IGF-I-receptor binding. Bovine CL were collected from a local abattoir and classified according to stage of diestrus based on anatomical characteristics. Corpora lutea from early, mid and late diestrus were each analyzed for the presence of IGFBP-3 by ligand blot analysis, and for RNA encoding IGFBP-3 by Northern blot analysis. Dissociated cells from mid-cycle CL were treated with IGF-I, IGFBP-2 or -3, or a combination of IGF-I and IGFBP-2 or -3. The effect of IGFBP-2 and IGFBP-3 on [(125)I] IGF-I binding to its receptor on CL plasma membranes also was investigated. IGFBP-3 protein and RNA expression were higher in early CL, compared to mid or late CL (p < 0.05). IGF-I stimulated P4 production in a dose-dependant manner (p < 0.05). IGFBP-2 and -3 blocked the stimulatory effect of IGF-I on P4 production (p < 0.05). Both IGFBP-2 and -3 inhibited [(125)I]-IGF-I binding to its receptor in a dose-dependant manner. These results demonstrate that IGFBP-3 protein and RNA are expressed predominantly during early diestrus in the bovine CL. Moreover, both IGFBP-2 and -3 can modulate IGF-I actions in the CL by interfering with binding of IGF-I to its receptor.     

Porcine insulin-like growth factor (IGF)-binding protein-3 elicits bi-phasic effects on IGF-I stimulated DNA synthesis in neonatal porcine skin fibroblasts

The present study was undertaken to investigate the effects of porcine IGFBP-3 on IGF-I stimulated DNA synthesis in neonatal porcine skin fibroblasts. IGF-1 stimulated DNA synthesis in skin fibroblasts in a concentration dependent manner. DNA synthesis was maximally stimulated by 5 to 20 fold at 5 nM IGF-I; half-maximal stimulation was observed at approximately 1 nM IGF-I. Co-incubation of IGFBP-3 with a maximally effective dose of IGF-I (10 nM) did not inhibit the stimulatory effects of IGF-I on DNA synthesis. In contrast, when IGFBP-3 at concentrations of 0 to 20 nM was co-incubated with 1 nM IGF-I, a bi-phasic dose response was observed with IGFBP-3 being inhibitory only at a 10 to 20 fold molar excess to IGF-I. Based on the approximately equal molar ratio of IGFBP-3:IGF-I present in the circulation of control and pST-treated pigs our results suggest that IGFBP-3 does not inhibit the mitogenic effects of IGF-I. In summary, these results indicate that the combination of IGFBP-3 with IGF-I optimizes mitogenic signalling via the type I IGF receptor and suggest that IGFBP-3 does not inhibit the effects of ST that are mediated by IGF-I.     

Ontogeny of hepatic bovine growth hormone receptors in cattle

A series of studies examined the binding characteristics and ontogeny of hepatic growth hormone binding sites in dairy bulls on d 2, 30, 180, and 365 of age. Binding of iodinated recombinant bovine growth hormone ([125I]rbGH) to liver membrane receptors was membrane protein-dependent. Receptors were considered growth hormone-specific, because physiological concentrations of bovine prolactin (bPRL) failed to displace [125I]rbGH from bovine hepatocyte membranes. Only 50% of [125I]rbGH was bound reversibly to hepatic microsomes. Addition of dithiothreitol (DTT) to the receptor-assay buffer increased the binding of [125I]rbGH to hepatic membranes in a time-dependent manner. Moderate concentrations of Ca++ and Mg++ in the receptor-assay buffer had no detectable effects on binding of [125I]rbGH to hepatic microsomes. In growing dairy bulls, specific binding of [125I]rbGH per milligram of membrane protein increased from 1.9 +/- 1.8% at d 2 to 14.1 +/- 1.8% at d 180 and then declined to 5.2 +/- 1.6% at d 365. Likewise, concentration of insulin-like growth factor (IGF)-I in serum was low during the 1st mo of age (d 2, 13.3 +/- 8.8 ng/ml; d 30, 9.7 +/- 8.8 ng/ml), but it became maximal at d 180 (151.0 +/- 8.8 ng/ml). Circulating concentrations of IGF-II increased linearly during the 1st yr of growth. Serum concentrations of GH, triiodothyronine, and thyroxine declined from 39.9 +/- 6.5, 2.7 +/- .2, and 75.4 +/- 4.6 ng/ml at d 2 to 16.5 +/- 6.5, 1.3 +/- .2, and 53.4 +/- 4.6 ng/ml at d 30, respectively, and remained low through 1 yr of age. Insulin concentration in serum did not change significantly with development. Results indicated that increasing concentrations of specific bGH receptors in the bovine liver may play a key role in regulating postnatal growth in cattle.     

Comparative study of the cardiovascular effects of losartan in normal and in water- and salt-depleted sheep

The cardiovascular effects of losartan, a non-peptidic angiotensin II (ANG II) receptor antagonist, were studied in sheep. Eight normotensive, conscious sheep were tested twice: first under normal conditions and second when subjected to water and electrolytic depletion (furosemide 5 mg/kg twice a day for 3 days). Intravenous injection of 30 mg/kg losartan lowered the mean arterial blood pressure (MABP) in both control and water- and electrolyte-depleted sheep alike. The maximal decrease in MABP was significantly greater in diuretic-treated sheep than in controls (20.0 +/- 2.7 vs 9.3 +/- 1.1 mmHg) and occurred earlier (8.0 +/- 3.3 min vs 12.1 +/- 2.9 min). The decrease in blood pressure was associated with tachycardia in both controls and diuretic-treated sheep (+5.5 +/- 1.8 vs +11.3 +/- 3.9 beats/min). The vasopressor response to 0.1 microg/kg ANG II administered 30 min after losartan was completely antagonized. Two hours after losartan administration, MABP was on the increase in all animals and ANG II receptor blockade was partially obliterated in control sheep. The more marked cardiovascular effects recorded in diuretic-treated sheep as compared to control animals were associated with an increased activation of the renin-angiotensin system (plasma renin concentration: 6.51 +/- 1.33 vs 1.42 +/- 0.37 ng angiotensin I/ml/hr).     

Insulin-like growth factor binding protein-3: its biological effect on bovine granulosa cells

This study was aimed at testing the hypothesis that insulin-like growth factor binding protein (IGFBP)-3 can modulate hormone-dependent differentiation of granulosa cells in vitro. Granulosa cells from small (1 to 5 mm) follicles were collected from cattle, cultured for 2 d in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 d in serum-free medium with follicle-stimulating hormone (FSH) (50 ng/ml), recombinant human IGF-I (0, 1.3, 4.0, or 13.3 nM), or recombinant human IGFBP-3 (0 to 4.26 nM). In one series of experiments, IGFBP-3 (0.53 and 2.13 nM) inhibited (51% to 92% decreases; P < 0.05) progesterone and estradiol production induced by 1.3 nM of IGF-I, but did not influence (P > 0.10) granulosa cell numbers or steroidogenesis in the absence of IGF-I. Only 4.26 nM of IGFBP-3 inhibited (by 35%) the increase in granulosa cell numbers induced by 1.3 nM of IGF-I. In another series of experiments, 13.3 nM of IGF-I, but not 4.0 nM of IGF-I, was able to completely overcome the inhibitory effect of 4.26 nM of IGFBP-3 on estradiol production. The increase in cell numbers induced by 4.0 and 13.3 nM of IGF-I was attenuated (P < 0.001) by 4.26 nM of IGFBP-3. In a third series of experiments, IGFBP-3 inhibited 125I-IGF-I binding to granulosa cells. These results indicate that IGFBP-3 has a pronounced inhibitory effect on IGF-I action in cultured bovine granulosa cells, and that this inhibitory effect is likely attributable to IGFBP-3 binding/sequestering IGF-I. Thus, IGFBP-3 may play a significant role in regulating granulosa cell proliferation and steroidogenesis during follicular development in cattle.     

Effect of weaning at different ages on serum insulin-like growth factor I (IGF-I), IGF binding proteins and serum in vitro mitogenic activity in swine.

We investigated the effects of weaning or fasting of 21- or 35-d-old swine by monitoring serum mitogenic activity, circulating insulin-like growth factor I (IGF-I) and its binding proteins using L6 myoblast bioassays, RIA and ligand blotting techniques. Serum samples were collected from 21- or 35-d-old animals just before and 36 h after weaning or fasting. Sera from 21- and 35-d-old weaned animals were not significantly altered in their ability to promote myoblast proliferation, whereas sera from 21- and 35-d-old fasted animals caused 29 and 21% decreases (P less than .05) compared with preweaning. The mitogenic activity of control serum was inhibited by serum from fasted animals but not by preweaned or weaned sera. Serum IGF-I levels were decreased 65 to 70% (P less than .05) with weaning or fasting at both ages. Unoccupied binding sites on circulating IGF binding proteins in the 155 kDa range decreased 18 to 19% with weaning at both ages and decreased 40% (P less than .05) with fasting at 21 d but only 17% at 35 d. Ligand blotting revealed that the 43 and 39 kDa IGF binding protein bands decreased with weaning and fasting at both ages, whereas the 29-kDa band increased with weaning and fasting. These data indicate that serum IGF-I and specific IGF binding protein bands decrease during weaning or fasting at 21 and 35 d of age. However, serum mitogenic activity did not always follow serum IGF-I levels.     

Effect of active immunization against growth hormone releasing factor on concentrations of somatotropin and insulin-like growth factor I in lactating beef cows.

Two experiments were conducted to determine the effects of immunoneutralization of growth hormone-releasing factor [GRF(1-29)-NH2] on concentrations of somatotropin (ST) and insulin-like growth factor I (IGF-I) in lactating beef cows. In Experiment 1, multiparous Hereford cows were immunized against 2 mg GRF(1-29)-(Gly)4-Cys-NH2 conjugated to human serum albumin (GRFi, n = 3) or 2 mg human serum albumin (HSAi, n = 3) at 52 +/- 1 d prior to parturition. Boosters (1 mg) were administered on days 12, 40 and 114 postpartum (pp). Serum samples were collected at 15-min intervals for 5 hr on days 18, 46 and 120 pp, followed by administration (IV) of an opioid agonist (FK33-824; 10 micrograms/kg) and an antagonist (naloxone; .5 mg/kg) at hours 5 and 7, respectively. A GRF-analog ([desamino-Tyr1, D-Ala2, Ala15] GRF (1-29)-NH2; 3.5 micrograms/kg) and arginine (.5 g/kg) were administered at hour 10 on days 47 and 121, respectively. Percentage binding of [125I]GRF (1:100 dilution of serum) 28 d after primary immunization was greater in GRFi (14.3 +/- 4.9) than in HSAi (.7 +/- .3) cows. Binding increased to 29.3 +/- 6.5% after first booster in GRFi cows. Episodic release of ST was abolished by immunization against GRF; concentration and frequency of release of ST were lower (P less than .05) in GRFi than in HSAi cows on all days pp. Concentrations of IGF-I were lower in GRFi than in HSAi cows throughout lactation. Serum ST failed to increase following FK33-824 or arginine in GRFi; however, ST increased after both compounds in HSAi cows. Concentrations of ST following GRF-analog were greater (P less than .05) in HSAi than in GRFi cows. Experiment 2 was conducted to determine if a lower dose of antigen and a single booster would be sufficient to lower ST and IGF-I in lactating cows. Multiparous Hereford and Angus cows were assigned to GRFi (n = 6) or HSAi (n = 6). Primary (1.2 mg) and booster (.5 mg) immunizations were administered -14 and 8 d from calving, respectively. Cows were restricted to 60% of recommended intake of energy during lactation in order to elevate concentrations of ST. Serum samples were collected at 15-min intervals for 6 hr on days 26, 50, 73, 90 and 109 pp. Two of six GRFi cows had binding less than 10% (1:1,000 dilution of serum) and were omitted from further analyses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantitative analysis of thiaminase activity in certain fish species.

Thiaminase I and II activity of Baltic herring, vendace, smelt and dace was measured. All four fish species were found to contain thiaminase activity. The amounts of thiaminase activity in mug of thiamine split per 100 g of fish tissue per hour at 37 degrees C were: in Baltic herring 115 +/- 60, in dace 11500 +/- 2050, in smelt 25 +/- 25 and in vendace 30+/- 15.     

Age dependence of the density of alpha-1 adrenergic receptors in liver cell membranes of domestic fowl]

Age dependence of alpha 1-adrenergic receptor number on hepatocytes of chickens was determined by incubation of liver cell membranes from chickens of different ages with 3H-prazosin. We showed, that there is a continuous and significant rise of alpha 1-adrenergic binding sites with increasing age of the chickens from 226 +/- 22 fmol/mg protein at the age of up to 4 weeks to 628 +/- 34 fmol/mg protein in laying hens. Changes in receptor affinity could not be registered. The KD-values ranged insignificantly from 0.34 +/- 0.03 nM to 0.50 +/- 0.06 nM in the different ages of chickens and amounted to 0.44 +/- 0.02 nM averagely. The classification of the binding sites for 3H-prazosin to the alpha 1B-subtype could be caused by the high sensitivity to chlorethylclonidine and by the low affinity for alpha A-adrenergic antagonists.     

Insulin-like growth factor (IGF)-I and -II and IGF binding proteins in serum and mammary secretions during the dry period and early lactation in dairy cows.

Concentrations of IGF-I and IGF-II, and IGF binding proteins (IGFBP) in serum and mammary gland secretions were surveyed during the dry period and early lactation of 30 Holstein cows. Although there was a threefold drop in the concentration of IGF-I in serum from the last week of the dry period to parturition (81 +/- 7 to 24 +/- 3 ng/ml, P less than .01), there was no significant change in serum IGF-II concentration during this period (150 +/- 17 vs 173 +/- 13 ng/ml, P greater than .05). Furthermore, a 57% increase in serum IGF-I was observed from the last week of lactation to the second week of drying off (100 +/- 5 to 157 +/- 8 ng/ml, P less than .05). Changes in serum IGF-II were not observed (126 +/- 11 vs 150 +/- 10 ng/ml, respectively; P greater than .05). Although IGF-I, IGF-II, and IGFBP concentrations in mammary secretions peaked 2 wk before parturition (2.95 +/- 1.1, 1.83 +/- .6, and 7.27 +/- .76 micrograms/ml, respectively), total output/quarter was highest in colostrum (394 +/- 119, 295 +/- 132, and 2,680 +/- 1,967 micrograms/quarter, respectively). Weekly milking of two individual quarters during the dry period did not affect (P greater than .05) IGF-I or IGF-II concentration (ng/ml) or total output (microgram/quarter) and milk yield in colostrum and milk (2 wk and 7 wk) compared with the ipsilateral quarter. The data support the hypothesis that IGF-I may be transported by the mammary gland epithelium. Furthermore, the secretion mechanisms of IGF-I, IGF-II, and IGFBP by the gland may be related to each other.     

Stability of vitamin E in blood and plasma from cattle, sheep, and pigs.

The stability of alpha-tocopherol concentrations in sheep, cattle, and pig blood and plasma stored at different temperatures was examined. For all species, the vitamin was stable for at least 6 days in plasma stored at -20 C, 4 C, and 25 C and in blood stored at 4 C and 25 C. For sheep and cattle, the vitamin was stable for at least 6 days in plasma stored at 37 C, but it was unstable in blood from all species stored at 37 C and in pig plasma stored at 37 C.