Binding Of 125I Research Articles

Characterization of ability of various substance P antagonists to inhibit action of bombesin

In the present study the ability of two classes of substance P (SP) antagonists to affect SP- and bombesin (Bn)-stimulated amylase release, as well as binding of 125I-Bolton-Hunter (BH)-SP and 125I-[Tyr4]Bn, was studied to determine their mechanism of action and the relationship between inhibition of the action of SP and the action of Bn. Four SP analogues ([D-Pro2,D-Phe7,D-Trp9]SP, [D-Pro2,D-Trp7,9]SP, [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP, and [D-Arg1,D-Trp7,9,Leu11]SP) and two SP-(4-11) analogues ([D-Pro4,D-Trp7,9]SP-(4-11) and [D-Pro4,D-Trp7,9,10]SP-(4-11)] did not stimulate amylase release when present alone but inhibited SP- and Bn-stimulated amylase release. For each SP analogue, inhibition was specific for peptides that stimulate release by interacting with SP or Bn receptors, whereas both SP-(4-11) analogues also inhibited cholecystokinin-stimulated amylase release. Each SP analogue's inhibition of Bn-stimulated amylase release was reversible. Each analogue inhibited binding of 125I-BH-SP with the same potency as it inhibited SP-stimulated amylase release and 125I-[Tyr4]Bn binding with the same potency as it inhibited Bn-stimulated amylase. In contrast, SP itself or SP-(4-11) itself did not inhibit Bn-stimulated amylase release or binding of 125I-[Tyr4]Bn. The relative affinities of the analogues for interacting with Bn and SP receptors differed. None of the analogues increased the dissociation of bound 125I-BH-SP or 125I-[Tyr4]Bn.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the high-affinity receptors on Swiss 3T3 cells which mediate the binding, internalization and degradation of the mitogenic peptide bombesin.

Bombesin and bombesin-related peptides such as gastrin-releasing peptide (GRP) stimulate DNA synthesis and proliferation of Swiss 3T3 cells in culture. We have used 125I-labelled [Tyr4]bombesin and 125I-labelled GRP to characterize and identify the receptors for these peptides on Swiss 3T3 cells. The binding of 125I-[Tyr4]bombesin, which retained full biological activity, was maximal between 20 and 30 min incubation at 37 degrees C, after which continued incubation led to a decline in cell-associated radioactivity. This decline was markedly slowed by the presence of lysosomal enzyme inhibitors. Specificity of the binding site was indicated by the competitive inhibition of binding by bombesin-related peptides, but not by unrelated peptides and growth factors. Scatchard analysis of binding data indicated a single class of high-affinity receptors. The calculated value for the dissociation constant (Kd) was 2.1 nM and each cell possesses approx. 240,000 receptors. Because [Tyr4]bombesin has no free amino group, 125I-GRP was used in chemical cross-linking studies. When disuccinimidyl suberate was used to covalently couple 125I-GRP to the cells, two major radiolabelled complexes were detected with molecular masses of approx. 80,000-85,000 and 140,000. The binding of 125I-[Tyr4]bombesin to the cells was pH-dependent with maximal binding at pH 6.5-7.5 and effectively no specific binding at pH values below 4.5. At 37 degrees C, cell-associated 125I-[Tyr4]bombesin quickly became resistant to removal by acidic buffers, suggesting its rapid transfer to an intracellular compartment. However, pre-incubation with unlabelled [Tyr4]bombesin did not induce down-regulation of bombesin receptors as measured by the subsequent binding of 125I-[Tyr4]bombesin. In contrast with the Swiss 3T3 cells, specific binding of 125I-[Tyr4]bombesin was not detectable in two cell lines which are biologically unresponsive to bombesin-related peptides.

Localization of angiotensin II receptor binding in rabbit brain by in vitro autoradiography.

Binding of 125I-[Sar1,Ile8] angiotensin II (AII) to sections of brains from both wild and laboratory rabbits was determined by in vitro autoradiography. In the forebrain, specific high density binding was observed in the olfactory bulb, organum vasculosum of the lamina terminalis (OVLT), subfornical organ, median eminence, lateral septum, median preoptic nucleus and hypothalamic paraventricular, supraoptic and arcuate nuclei. In the midbrain, binding of the radioligand was observed in the interpeduncular and parabrachial nuclei, in the locus coeruleus, and ventrolateral pons. In the hind brain, there was dense binding of 125I-[Sar1,Ile8] AII to the nucleus of the solitary tract (NTS) and to both rostral and caudal parts of the reticular formation of the ventrolateral medulla oblongata. Weaker specific binding of the radioligand to the molecular layer of the cerebellum, to the nucleus of the spinal trigeminal tract, dorsal motor nucleus of the vagus, area postema, and to a band of tissue connecting the NTS to the ventrolateral medulla was also observed. Binding of the ligand to circumventricular organs such as the OVLT, subfornical organ, and median eminence suggests that these are sites in the brain of the rabbit at which blood-borne AII may exert influences on the central regulation of fluid balance and pituitary hormone secretion, although AII of neuronal origin could also act at these sites. Binding of the radioligand in several other brain regions suggests that angiotensin II of cerebral origin may be involved in a number of different aspects of brain function in the rabbit. The finding of dense binding in the NTS and ventrolateral medulla, which are involved in autonomic activity and are also sites of catecholamine-containing neurons, raises the possibility of angiotensin interaction with these neurons and involvement in autonomic function.

Receptors for calcitonin gene-related peptide on the rat liver plasma membranes

Specific binding sites for calcitonin gene-related peptide (CGRP) were identified in the rat liver plasma membrane. The binding of 125I-[TyrO]rat CGRP to rat liver plasma membrane was time dependent, saturable and reversible. Scatchard analysis of the data revealed a single class of binding sites with apparent dissociation constant of 260.8 pM and a maximal binding capacity of 26.6 fmol/mg of protein. Rat, chick, and human CGRP and their synthetic analogues inhibited label binding in a dose-dependent manner with relative potencies as follows; chick greater than rat greater than human greater than [TyrO]rat CGRP. Salmon, human and [Asu1'7]eel calcitonin also inhibited label binding but only at higher concentrations. These results clearly indicate the presence of specific binding sites for CGRP in rat liver plasma membrane and suggest that CGRP has possible biological actions on the rat liver.

Inability to detect transferrin receptors on P. falciparum parasitized red cells.

The mechanism by which P. falciparum takes up iron from transferrin has been explored. Binding of 125I labelled transferrin to parasitized red cells at 37 degrees C is two-fold greater than to control cells; at 0 degrees C there is no significant difference. The binding is non-specific as judged from the following: it is not saturable; it is not limited to transferrin as lactoferrin (which has iron binding domains) and bovine serum albumin (which does not) also bind in excess to parasitized red cells. A transferrin receptor complex could not be demonstrated when parasitized red cells, to which 125I transferrin was bound, were solubilized in Triton X100. Previous observation showed that uptake of transferrin iron by parasitized red cells is not accompanied by equimolar uptake of transferrin protein. We therefore suggest that nonspecifically bound transferrin is endocytosed, that the protein is degraded and the iron selectively retained.

Localization of specific binding sites for bombesin in the canine gastrointestinal tract

The goal of these studies was to determine the tissue and cell types possessing specific binding sites for bombesin/gastrin-releasing peptide in the canine gastrointestinal tract. Monoiodinated, biologically active (Tyr-4)-bombesin 14 (100 pM) was applied to sections of canine gut and localized using quantitative autoradiography. The highest density of bombesin/gastrin-releasing peptide specific binding sites occurred over endocrine cells in the antral mucosa. Specific binding sites were also found on the circular muscle layer of the gastric fundus, gastric antrum, and ileum, on longitudinal muscle of the gastric fundus and antrum, and on neuronal elements in the myenteric plexus in the gastric fundus, antrum, and small intestine. No evidence for specific binding of 125I-(Tyr-4)-bombesin 14 was found in sections of canine esophagus, gastric cardia, gallbladder, pancreas, or colon. These results suggest sites of direct action of bombesin and endogenous gastrin-releasing peptide for gastrin release and gastrointestinal motility.

Angiotensin receptor binding in human hypothalamus: autoradiographic localization

Binding of 125I-[Sar 1,Ile 8]angiotensin II in the human hypothalamus was mapped by in vitro autoradiography carried out on frozen sections of hypothalamus from two human brains. Regions showing the greatest specific binding of this radioligand were the organum vasculosum of the lamina terminalis, median preoptic nucleus, subfornical organ, median eminence, arcuate nucleus and paraventricular nucleus. These regions may be sites of angiotensin II receptors involved in the regulation of blood pressure, fluid balance and pituitary hormone secretion.

Trichloroacetic acid (TCA) precipitability of 125I in the blood of mice fed 125I

A recent study showed that after feeding 125I-labelled proteins or free 125I to mice, as much as 40% of total radioactivity in the circulation precipitated upon mixing whole blood with 10% trichloroacetic acid. We examined this potential limitation to the use of radiolabelled tracers for studies on intestinal digestion of proteins and protein uptake, and identified its mechanism. BALB/c mice were gavage-fed or injected intravenously (i.v.) with Na 125I. Blood obtained at 15 min was added directly to 10% trichloroacetic acid (TCA) or was processed to obtain serum or plasma. On mixing with 10% TCA, 25–35% of the radioactivity in whole blood was precipitated; less than 2% of the radioactivity in plasma or serum was precipitated. In vitro studies identified hemoglobin as the primary carrier protein participating in this reaction. If hemoglobin was replaced by methemoglobin or cyanomethemoglobin, then the reaction with 125I did not occur, suggesting that iron in the heme group may be the site for 125I binding and that iron must be in its reduced or ferrous form (Fe 2+). The administration of non-radioactive NaI in vivo or its addition to reaction mixtures in vitro completely inhibited the precipitation of 125I by hemoglobin in the presence of TCA. Thus the addition of non-radioactive iodide to TCA stock solutions may effectively prevent non-specific binding of 125I to free hemoglobin released unintentionally during venipuncture or at other stages of blood processing.

Functional and biochemical characterization of B-cell differentiation factor (BCDF) produced by an HTLV-I-transformed human T-cell clone and demonstration of specific binding of the factor to a BCDF responsive cell line

We have previously described YA2, a human T-cell clone that secretes B-cell differentiation factor (BCDF) but not B-cell growth factor (BCGF). The BCDFs secreted by YA2 and HTLV-I-transformed YA2 (TYA2) were functionally similar in their ability to stimulate Ig secretion by Staphylococcus aureus Cowan strain I-activated B cells and IgM secretion by SKW6.4 cells. In addition, they were biochemically similar with a MW of 30 kDa by high-performance liquid chromatography (HPLC) sieving, and a p I of 6.0–6.8 by isoelectric focusing. The BCDF activity was not blocked by antibodies to interleukin 2 and BCGF. BCDF was purified from TYA2 supernatant by sequential media protein immunoadsorption, flat bed isoelectric focusing, HPLC TSK 2000 sieving, and repeated immunoadsorption and was then iodinated. The iodinated material had functional BCDF activity and migrated to a single band at MW 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at p I of 6.8 by polyacrylamide gel isoelectric focusing. 125I-BCDF purified in this manner bound specifically to a BCDF-responsive cell line and not to phytohemagglutinin-activated T cells. 125I binding to the BCDF-responsive cell line was competitively inhibitable by the addition of cold BCDF. Thus we have purified and characterized a factor with BCDF activity and demonstrated that this factor binds specifically to a BCDF-responsive cell line.

Identification of an Fc receptor for IgG1 and IgG2 on guinea-pig polymorphonuclear leukocytes

Ovalbumin (OA)-complexed guinea-pig IgG1 and IgG2 antibodies were found to bind to homologous polymorphonuclear leukocytes (PMNs). As these bindings are assumed to be mediated by certain Fc receptors (FcRs) for IgG1 and IgG2, the variety and properties of the FcRs on the cells were investigated by the use of two monoclonal antibodies to guinea-pig macrophage FcRs which were prepared by Shimamura T. et al., 1987 ( Molec. Immun. 24, 67–74): VI A2 IgG1 to the FcR for IgG1 and IgG2 (FcR 1.2) and VII A1 IgG1 to the FcR for IgG2 (FcR 2). PMNs were shown to bind the Fab' of VI A2 IgG1 (VI A2 Fab') by flow cytofluorometry, suggesting that the cells possess a certain FcR which cross-reacts antigenically with macrophage FcR 1,2. In fact, VI A2 Fab' inhibited completely the binding of OA-complexed IgG1 antibody to the cells. When the FcR was isolated by affinity chromatography on the F(ab') 2 of VI A2 IgG1 coupled to Sepharose, it gave a 55,000 mol. wt band on sodium dodecylsulfate-polyacrylamide gel electrophoresis, as in the case of macrophage FcR 1,2. The number of the FcR molecules per PMN cell was estimated to be 2 × 10 4 by measuring the binding of 125I–VI A2 Fab'. The binding of OA-complexed IgG2 antibody to PMNs was also inhibited with VI A2 Fab', but partially. This finding indicates that the FcR bound by VI A2 Fab' may be an FcR 1,2 which is able to bind both OA-complexed IgG1 and IgG2 antibodies, and also that PMNs possess another FcR, namely FcR; which binds IgG2 antibody alone. The Fab' of VII A1 IgG1 (VII A1 Fab'), on the other hand, did not exhibit any inhibitory activity on the bindings of OA-complexed IgG1 and IgG2 antibodies to PMNs. Since no evidence indicating the binding of VII A1 Fab' to PMN cells was obtained by flow cytofluorometry, the FcR 2 of PMNs may be antigenically different from its macrophage counterpart. In conclusion, these results indicate that two distinct types of FcR for IgG isotypes exist on guinea-pig PMN cells: FcR 1,2 similar to macrophage FcR 1,2, and FcR 2 distinct from macrophage FcR 2.

Alteration of EGF-receptor binding in human breast cancer cells by antineoplastic agents

Polypeptide growth factors bind to membrane receptors on human breast cancer cells and stimulate cell proliferation, suggesting that they may be important in growth regulation. Inhibition of the stimulatory effects of these factors might result in antineoplastic activity. Since cytotoxic drugs have been shown to alter cell membrane characteristics, we have examined the effects of a variety of antitumor drugs on the binding of epidermal growth factor (EGF) to the membrane receptor of human breast cancer cells. Twenty-four standard or investigational cytotoxic drugs were screened at a concentration of one-tenth the achievable peak plasma level for their ability to inhibit binding of 125I -EGF to its receptor in MCF- 7 human breast cancer cells. Although at this concentration statistically significant inhibition of binding was observed with 11 drugs, the maximum inhibition observed was only 27%. Five agents, representing classes of drugs with different modes of action, were then studied in more detail. Of these, preincubation with 5-fluorouracil, 4-hydroperoxy-cyclophosphamide and doxorubicin inhibited MCF- 7 colony formation in a dose-dependent manner, but these drugs had no effect on EGF-binding even at a concentration of 10 times the peak plasma level. Preincubation of cells with vinblastine and cisplatin, however, resulted in both reduced colony survival and a parallel reduction in EGF receptor binding. Membrane integrity, as measured by trypan blue exclusion, was not altered. Scatchard analysis of EGF binding demonstrated that the major effect of cisplain was a reduction in binding affinity. We conclude that cisplatin and vinblastine at high concentrations can inhibit the binding of EGF to human breast cancer cells offering an additional possible mechanism for their antiproliferative activity.

Solubilization and characterization of guinea-pig pancreatic somatostatin receptors.

The solubilization of somatostatin receptors from guinea-pig pancreas by different non-denaturing detergents was investigated after stabilization of the receptors by prior binding of 125I-[Tyr11]somatostatin or its analogue 125I-[Leu8,DTrp22,Tyr25]somatostatin 28, to pancreatic plasma membranes. The somatostatin-receptor complexes were solubilized in a high yield by Zwittergent 3-14 (3-[tetradecyldimethylammonio]-1-propanesulfonate), a zwitterionic detergent. Other detergents, digitonin, Triton X-100, Chaps (3-[cholamidopropyldimethylammonio]-1-propanesulfonate) and octyl beta-D-glycopyranoside, achieved only partial solubilization. The recovery of receptor complexes was increased by glycerol. In order to characterize solubilized somatostatin-receptor complexes, membranes receptors were covalently labelled using N-5-azido-2-nitrobenzoyloxysuccinimide as cross-linking reagent before solubilization. Gel filtration chromatography analysis resulted in the identification of a major protein component of apparent Mr = 93,000 which interacted with the two radioligands. In addition, a similar component of Mr = 88,000 was characterized after analysis by SDS-PAGE of membrane receptors covalently cross-linked with 125I-[Leu8,DTrp22,Tyr25]somatostatin 28 by different heterobifunctional reagents: N-5-azido-2-nitrobenzoyloxysuccinimide, N-hydroxysuccinimidyl 4-azidobenzoate, N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate. Optimal cross-linking results were obtained with N-5-azido-2-nitrobenzoyloxysuccinimide. The solubilized somatostatin-receptor complex was adsorbed to wheat-germ agglutinin-agarose column and eluted by specific sugars. We concluded that the guinea-pig pancreatic somatostatin receptor in the membrane and in the non-denaturing detergent solution behaves as a protein monomer of apparent Mr approximately 85,000-90,000. The somatostatin receptor is a glycoprotein which contains complex-type carbohydrate chains.

Bombesin binding and biological effects on pancreatic acinar AR42J cells

The effects of bombesin on amylase release and the receptor binding of 125I-[Tyr4]bombesin in the rat pancreatic acinar carcinoma cell line AR42J were examined. Bombesin-like peptides stimulated amylase release from AR42J cells in a dose-dependent manner; a maximal 2-fold stimulation occurred at a bombesin concentration of 300 pM. Binding of 125I-[Tyr4]-bombesin to AR42J cells was specific, saturable and temperature dependent. The relative potencies with which various structurally related peptides stimulated amylase release correlated well with their relative abilities to compete for the bombesin receptor.

Binding of rabies virus to purified Torpedo acetylcholine receptor

The binding of 125I- and 35S-labeled rabies virus (CVS strain) to affinity-purified acetylcholine receptor from Torpedo electric organ was demonstrated. The binding of rabies virus to the acetylcholine receptor increased with increasing receptor concentration, was dependent on the pH of the incubation medium, and was saturable with increasing virus concentration. Binding of radioactively labeled virus was effectively competed by unlabeled homologous virus particles. Binding of 35S-labeled rabies virus to the AChR was inhibited up to 50% by α-bungarotoxin and up to 30% by (+)-tubocurarine but was not affected by atropine. These results demonstrate direct binding of rabies virus to a well-defined neurotransmitter receptor, namely the acetylcholine receptor and indicate that at least a portion of the virus interaction occurs near the acetylcholine binding site on the receptor. These findings support the hypothesis that the acetylcholine receptor may serve as a rabies virus receptor in vivo.

Different ionic requirements for somatostatin receptor subpopulations in the brain

Two populations of brain somatostatin (SS) receptors, one with high affinity for the somatostatin octapeptide analogue SMS 201-995 (SS 1 type) and one poorly sensitive to this analogue (SS 2 type) have been characterised in regard to their ionic requirements using two radioligands, the iodinated Tyr 3 derivative of the octapeptide SS analog SMS 201-995 and the iodinated [Tyr 11]-SS. Specific binding of 125I-[Tyr 11]-SS to rat cortex membrane homogenates can be increased by approximately 180% in presence of 5 mM Mg 2+. The increase in number of binding sites seen by Mg 2+ is not accompanied by a marked increase in affinity for SS but for SMS 201-995: the low affinity binding for SMS 201-995 seen in absence of Mg 2+ is replaced in part by higher affinity binding in presence of these ions. SMS 201-995 sensitive SS 1 receptor subpopulation measured with 125I-204-090, a specific ligand for SS 1 subpopulation, is massively increased in presence of Mg 2+. However, SMS 201-995 insensitive SS 2 receptor population measured with 125I-[Tyr 11]-SS in presence of excess SMS 201-995 is unchanged in presence of Mg 2+. The Mg 2+-dependency can also be observed with autoradiography for extra cortical, i.e. hippocampal, brain SS receptors. 120 mM Na + does not affect the total brain SS receptor population, but reduces the specific binding of SS 1 receptors and increases that of SS 2 receptors. Therefore, the rat brain, in particular the cortex, possesses a SMS 201-995-sensitive, Mg 2+-dependent SS receptor subpopulation (SS 1) as well as a SMS 201-995-insensitive, Mg 2+-independent SS population (SS 2).

β-Adrenergic control of motility in the rat colon. II. Proportions of β1- and β2-adrenoceptors identified with 125I-(−)pindolol binding

The specific binding of 125I-(−)pindolol to membranes prepared from rat colon displayed high affinity (Kd = 75 ± 3 pM) and relatively low capacity (Bmax = 48 ± 3.8 fmol/mg protein). Furthermore, the binding was reversible and possessed properties expected of a β-adrenoceptor. Thus, for example, the potency of agonists in displacing 125I-(−)pindolol was, in decreasing order, isoproterenol > epinephrine > norepinephrine, and the negative isomer of propranolol was two orders of magnitude more potent than the positive isomer. Displacement of specific binding by the β1-adrenoceptor antagonist pafenolol and the β2-adrenoceptor antagonist ICI 118.551 revealed that the colon preparation possessed a heterogenous β-adrenoceptor population. Analysis of the inhibition curves using computerassisted curve fitting suggested that the sites consisted of a small (14%–21%) β1-adrenoceptor population and a large (79%–86%) β2-adrenoceptor population. The coexistence of β1- and β2-adrenoceptors is discussed in relation to evidence of a functional linkage of both subtypes with colon motility.

Calcium-dependence of somatostatin binding to receptors

Binding of 125I-[Tyr 11] somatostatin (S14) and 125I-[Tyr 1]S14 has been studied in pancreatic acini and cerebral cortex. Ca 2+-dependence of somatostatin binding to receptors was observed only with the highly non degradable iodinated analog 125I-[Tyr 11] somatostatin but not with 125I-[Tyr 1] somatostatin. The inhibitory activity of S14 on secretin-stimulated cAMP cellular content was decreased when Ca 2+ concentration in the medium was reduced to 30 nM, indicating that the Ca 2+ dependence of somatostatin binding to receptors is also present with the native peptide.

Rapid sequestration and degradation of somatostatin analogues by isolated brain microvessels.

Somatostatin (SRIF) is a putative peptide neurotransmitter that may interact with brain capillaries following neurosecretion of the peptide. The present studies investigate the binding and metabolism of SRIF analogues in isolated bovine brain microvessels. 125I-[Tyr1]SRIF was rapidly degraded by capillary aminopeptidase with a half-time of approximately 3 min at 23 degrees C. The microvessel aminopeptidase had a low affinity and high capacity for the peptide, Km = 76 microM and Vmax = 74 nmol min-1 mgp-1. 125I-[Tyr11]SRIF was converted to free iodotyrosine at a much slower rate, presumably by a lower-activity endopeptidase. 125I-[Try11]SRIF was rapidly bound by microvessels, whereas another basic peptide, [Tyr8]bradykinin, or an acidic peptide, CCK8, or a neutral peptide, leucine enkephalin, were bound to a considerably less extent. The binding of 125I-[Tyr11]SRIF to the capillaries was nonsaturable up to a concentration of 1 microgram/ml of unlabeled peptide, and the binding reaction was extremely rapid, reaching equilibrium within 5 s at either 0 degrees C or 37 degrees C. Approximately 20% of the SRIF bound by the microvessels was resistant to acid wash and presumably represented internalized peptide. In addition, the 125I-[Tyr11]SRIF bound rapidly to the endothelial cytoskeleton remaining after a 1% Triton X-100 extraction of the microvessels. The peptide-cytoskeletal binding reaction was nonsaturable up to 1 microgram/ml of unlabeled [Tyr11]SRIF, but it was inhibited by 0.5% polylysine or 0.8 M KCl and was stimulated by 1 mM dithiothreiotol. These studies suggest that brain microvessels rapidly sequester and degrade SRIF analogues and that this may represent one mechanism for rapid inactivation of the neuropeptides subsequent to neurosecretion.

Tumor promoter inhibition of cellular binding of somatostatin

Tumor promoting phorbol esters inhibited the binding of 125I-[Tyr 11] somatostatin to isolated acinar cells from guinea-pig pancreas. Maximal inhibition reached 69.7 ± 5 % at 1 μM TPA. Receptor affinity was decreased by 2.5-fold without change in binding capacity. The ability of TPA in inhibiting somatostatin binding was decreased in 30 nM Ca 2+ medium, abolished at 4°C or in a membrane preparation. The effect of caerulein, a secretagogue which also caused loss of binding, and that of TPA were not additive. We concluded that TPA inhibits somatostatin binding not by binding directly at the active site of somatostatin receptor. TPA may act at a later point than caerulein via a similar pathway to modulate somatostatin receptor affinity.

Binding and internalization of lysosomal enzymes by primary cultures of rat glia.

Highly purified cultures of rat astrocytes and oligodendrocytes were examined for their ability to bind and internalize lysosomal enzymes. Astrocytes displayed a saturable uptake of beta-glucosidase and beta-galactosidase. The uptake was specifically inhibited by mannose-6-phosphate but not by several other sugars or sugar phosphates, indicating that the process was mediated by mannose-6-phosphate receptors. When cells were allowed to take up 125I-beta-glucosidase for 1 hr at 37 degrees C and subcellular organelles were isolated, the enzyme was shown to comigrate with a lysosomal organelle marker enzyme, suggesting that the enzyme was targeted to lysosomes. Astrocyte receptors were probed directly by binding of 125I labeled beta-glucosidase to astrocyte membranes at 4 degrees C. Binding was saturable and competitively inhibited by mannose-6-phosphate. In contrast to the astrocytes, cultured oligodendrocytes showed no specific binding or uptake of the lysosomal enzymes. Immunocytochemical staining of mixed glial cultures supported the biochemical data; only the astrocytes stained positive with anti-mannose-6-phosphate receptor antibodies.