igh sialic acid content slows prourokinase turnover in rabbits.

The clearance of natural and recombinant prourokinase (proUK) from the blood of rabbits was studied by means of a double-isotope method which allowed the differential removal of two distinct proUK species to be monitored when simultaneously administered to an individual animal. In initial experiments, proUK expressed in different cell lines contained between 0 and 2.5 molecules of sialic acid per molecule of protein. A slight trend toward slower clearance of proUK with higher sialic acid content was observed but rate differences were not statistically significant. Recombinant proUK produced in CHO cells grown in flow reactors, contained unusually high levels of sialic acid in excess of 3 moles/mole protein. Controlled exposure to immobilized neuraminidase was used to remove sialic acid from this protein in defined amounts. The clearance of the parent material was biphasic with average alpha and beta half-lives of 1.7 min and 16.7 min respectively. The AUC of the parent material was only slightly lowered upon removal of 30% of the original sialic acid. Species with 60% or 90% removal of sialate were much more rapidly cleared from the circulation respectively yielding AUCs equal to 56% and 41% of that observed with the parent material. Thus proUK containing 2.5-3.5 sialic acid molecules per molecule of protein turned over significantly more slowly in rabbits than did less sialylated proUK. The clearance rate was relatively insensitive to sialic acid content between 0 and 1.5 sialic acid residues per proUK molecule.

Differential incorporation of sulfate into the chondroitin chain and complex carbohydrate chains of human thyroglobulin: studies in normal and neoplastic thyroid tissue.

Human thyroglobulin (TG) is one of a growing number of glycoproteins that are known to contain sulfate. Among these TG is unusual because it contains sulfate on both its asparagine-linked complex carbohydrate units and its single chondroitin 6-SO4 unit. We incubated tissue fragments prepared from normal and neoplastic thyroid tissue with [35S]sulfate to study the incorporation of sulfate into these two types of carbohydrate acceptor sites. Incubation conditions (0.1 mM sulfate for 16 h) were selected that maintained linear incorporation of [35S]sulfate and retention of more than 90% of iodinated TG in the tissue. Enzyme susceptibility was used to determine incorporation into the complex carbohydrate units (endoglycosidase-F) and the chondroitin 6-SO4 unit (chondroitin ABC lyase). In a representative experiment, 29.8% of the incorporated sulfate was found in the chondroitin 6-SO4 unit during the first hour of incubation. This increased progressively to 72.5% in the chondroitin unit during the incubation period from 8-16 h. A reciprocal decrease occurred in the proportion of sulfate incorporated into the complex carbohydrate units. TG released into the medium and retained in the tissue had the same ratio of sulfate in the two types of carbohydrate units. Neoplastic thyroid tissue incorporated more [35S]sulfate into TG than normal thyroid tissue from the same patient. Neoplastic and normal tissue differed further in the ratios of sulfate incorporated into the two types of carbohydrate units. We conclude that the incorporation of sulfate into the two types of sulfate-containing carbohydrate units of TG does not occur in a fixed ratio and that this differential incorporation of sulfate does not appear to be related to its release from the tissue.

Metabolic labeling of human thyroglobulin with [35S]sulfate: incorporation into chondroitin 6-sulfate and endoglycosidase-F-susceptible carbohydrate units.

To study the incorporation of sulfate into thyroglobulin, human thyroid tissue was incubated with [35S]sulfate. Labeled thyroglobulin was purified by ammonium sulfate precipitation, gel exclusion chromatography, and equilibrium density gradient centrifugation, the last of these specifically to remove proteoglycans. [35S]Sulfate was found in thyroglobulin with low density, indicating that sulfate was incorporated into newly synthesized molecules before their iodination. Chondroitin ABC lyase and chondroitin AC lyase released equal amounts of [35S]sulfate, demonstrating the presence of chondroitin units, and TLC showed this to be predominantly chondroitin 6-sulfate. Additional [35S]sulfate was released by endoglycosidase-F (Endo-F), but not by Endo-H. The Endo-F-sensitive sulfate-labeled complex carbohydrate units were heterogeneous, one fraction passing through a Concanavalin-A-Sepharose column without delay and another fraction showing weak affinity for the lectin. More than 90% of the incorporated [35S]sulfate was accounted for by the chondroitin ABC lyase and Endo-F-sensitive fractions. Thus, human thyroglobulin contains sulfate in at least three types of carbohydrate units, 1) chondroitin 6-sulfate units, 2) complex units with no affinity for Concanavalin-A (tri- or tetraantennary forms), and 3) complex units with weak affinity for Concanavalin-A (biantennary forms).

Ectopic growth hormone-releasing factor stimulates growth hormone secretion in the urethane-anesthetized rat in vivo.

The in vivo bioactivity of ectopic growth hormone-releasing factor (GHRF) was examined in estrogen-primed, urethane-anesthetized male rats bearing intracarotid catheters. Ectopic GHRF was isolated from a carcinoid tumor metastatic to the liver from a patient with gigantism and elevated plasma growth hormone (GH) levels and was partially purified by reverse phase high performance liquid chromatography for intracarotid injection. Ectopic GHRF elicited a promt rise in plasma GH which peaked at 5 min. The time course of the response resembled that to prostaglandin E1, a known potent direct stimulator of GH secretion in vivo. The effect of ectopic GHRF was dose related in the range of 10-100 U/rat which represents approximately 30x the effective in vitro dose range/10(5) cells in rat adenohypophyseal cell cultures. The in vivo response to ectopic GHRF was completely blocked by the simultaneous intracarotid administration of the GH release inhibitory peptide, somatostatin. These findings lend further support for a role of GHRF from human tumors as a potent physiological stimulator of GH release.

Release of somatostatin-like immunoreactivity from incubated rat hypothalamus and cerebral cortex. Effects of glucose and glucoregulatory hormones.

Somatostatin (SRIF) is localized in the hypothalamus, extrahypothalamic brain, and throughout the gastrointestinal tract. Release of gastrointestinal SRIF-like immunoreactivity (SRIF-LI) is under nutrient regulation but the effect of nutrients on neural SRIF-LI is unknown. The present studies examined the effects of glucose uptake and metabolism and hormones influencing glucose disposition on SRIF-LI release from medial basal hypothalamus (MBH) and cerebral cortex (Cx) incubated in Krebs-Ringer bicarbonate containing bacitracin. After a preincubation to achieve stable secretion, tissues were incubated for 20 min in 14 mM glucose (basal) and then, for 20 min in fresh medium with test materials. MBH SRIF-LI release was inversely related to medium glucose concentration with release in the absence of glucose (235+/-42 pg/MBH per 20 min) more than five times that in the presence of 25 mM glucose (46+/-4 pg/20 min). In the presence of 14 mM glucose MBH SRIF-LI release was stimulated above basal by agents interfering with glucose uptake including 3-O-methyl-d-glucose (42 mM; 70+/-5 vs. 42+/-3 pg/20 min, P < 0.05), phlorizin (50 mM; 351+/-63 vs. 29+/-2 pg/20 min, P < 0.001) or cytochalasin B (20 muM; 110+/-7 vs. 22+/-2 pg/20 min, P < 0.001). Inhibition of glucose metabolism by 2-deoxy-d-glucose resulted in dose-related stimulation of MBH SRIF-LI release (maximal at 28 mM; 201+/-28 pg/20 min vs. 32+/-4 pg/20 min, P < 0.001). Viability of MBH was unimpaired by incubation in the absence of glucose or following exposure to 2-deoxy-d-glucose as determined by retention of SRIF-LI responsiveness to stimulation by potassium (60 mM) or neurotensin (5 muM). In contrast, Cx SRIF-LI release was slightly inhibited by decreases in medium glucose and unaffected by inhibition of glucose uptake or metabolism. These results provide evidence for nutrient regulation of MBH but not Cx SRIF-LI release and may explain inhibition of growth hormone seen in the rat in response to hypoglycemia. Insulin (10 nM-1 muM) stimulated MBH but not Cx SRIF-LI release while glucagon was without effect. Our previous demonstration that MBH SRIF-LI release was stimulated by somatomedin-C, but not insulin at physiologic concentrations, is consistent with an action of insulin through the somatomedin-C receptor at the doses studied. Our studies indicate a regional specificity for the control of SRIF secretion within the brain and suggests the possibility of a role for hypothalamic SRIF in metabolic regulation.