Tracer Degradation Research Articles

3H]Angiotensin II binding to basolateral membranes from rat proximal renal tubule: effect of sodium intake and captopril

Basolateral membranes were prepared from rat renal cortex by density gradient centrifugation. Their purity was confirmed by electron microscopy and by marker enzyme enrichment. The basolateral membrane preparation was shown to be derived predominantly from the proximal renal tubule by measurement of hormone-stimulated adenylate cyclase; marked stimulation of adenylate cyclase was found with parathyroid hormone, but not with isoprenaline, antidiuretic hormone or calcitonin. A single class of specific high-affinity [3H]angiotensin II-binding site was identified in the basolateral membrane preparation which, after correction of results for tracer degradation, showed equilibrium dissociation constant of 0.23 nmol/l and binding site concentration of 485.8 fmol/mg protein. Binding sites for [3H]angiotensin II were measured in basolateral membranes prepared from rats fed diets with a low, normal or high sodium content. A trend of increased binding site density with reduced sodium intake was found which did not reach statistical significance. No effect on affinity was demonstrated. Treatment of rats on a low-sodium diet with captopril (500 mg/l drinking water) caused a significant reduction in binding site density; no effect on affinity was demonstrated. These findings suggest that the density of angiotensin II receptors at this site is altered by the activity of the renin-angiotensin system.

Albumin absorption by canine bronchial epithelium.

Albumin concentrations in airway surface liquid are low compared with plasma. To investigate the mechanisms that generate albumin gradients across airway epithelia, we have investigated whether active albumin absorption is a feature of bronchial epithelia. Freshly excised canine bronchi were mounted in Ussing chambers and short-circuited. Permeability coefficients of 14C-labeled canine albumin (Palb) were measured in the mucosal-to-submucosal (M----S) and submucosal-to-mucosal (S----M) directions in conductance-matched tissues. Mean steady-state values for Palb in the absorptive (M----S) direction (5.97 +/- 1.89 x 10(-7) cm/s) were significantly greater than rates in the S----M direction (1.09 +/- 0.41 x 10(-7) cm/s). Simultaneous measurements detected no asymmetry of transport of the fluid phase marker [3H]inulin. Gel filtration chromatography demonstrated that the majority of the radiolabel released into the submucosal bathing solution represented albumin fragments. Albumin fragments per se were not transported because no asymmetries in permeabilities of albumin fragments isolated from spontaneous degradation of tracer were detected. Decreasing the temperature of the bathing solution from 37 to 4 degrees C completely inhibited net albumin absorption. [14C]albumin transport was saturated by addition of high concentrations of unlabeled albumin (estimated Michaelis constant = 1.6 x 10(-3) M). These results demonstrate that albumin is absorbed by a low-affinity process that may contribute to the maintenance of low albumin concentrations in secretions.

Specific binding of atrial natriuretic peptide to rat mesenteric artery: effect of calcium and 5-guanylylimidodiphosphate.

1. High-dissociation equilibrium constant (20 pmol/1, receptor number 17 fmol/mg) and low-dissociation equilibrium constant (10 nmol/1, receptor number 5 nmol/mg) affinity binding sites for atrial natriuretic peptide have been identified in membrane preparations from rat mesenteric artery. 2. Tracer degradation was corrected for mathematically and binding data were analysed by a non-linear computer technique. 3. Non-atrial natriuretic peptide vasoactive hormones, angiotensin II, vasopressin and bradykinin, did not compete for the high-affinity site. Related peptides with either C- or N-terminal amino acids missing still competed, while peptides without an intact disulphide bridge did not compete. 4. Neither the addition nor the removal of calcium affected the affinity or density of binding sites. Also the non-hydrolysable guanosine triphosphate analogue 5-guanylylimidodiphosphate had no effect on either affinity or number of sites. 5. These results indicate the presence of a specific high-affinity vascular receptor for atrial natriuretic peptide which could interact with the hormone under physiological conditions. The mechanism of binding is uncertain but is unlikely to involve calcium- or guanosine triphosphate-associated regulatory sites.

The use of reversed-phase high-performance liquid chromatography to detect proteolytic activity from human pancreas in a radioimmunoassay for corticotrophin-releasing factor.

Degradation of tracer during a radioimmunoassay (RIA) can result in false-positive concentrations of immunoreactivity being reported in a biological sample. A technique has been developed using reversed-phase high-performance liquid chromatography (HPLC) to detect proteolytic degradation of corticotrophin-releasing factor-41 (CRF-41) during incubation with tissue extracts under RIA conditions. Human pancreatic tissue was extracted in HCl or urea and incubated with 125I-labelled CRF-41 at neutral pH for 18 h. When samples were analysed by HPLC and fractions counted for radioactivity, tracer was extensively degraded. Heating extracts at 85 degrees C or adding lima bean trypsin inhibitor to the medium prevented degradation. Pancreatic tissue extracted in HCl was analysed by gel filtration and HPLC, and fractions were subjected to RIA for CRF-41. A peak of immunoreactivity was detected by both chromatographic methods. However, when this material was incubated with tracer and analysed by HPLC, the tracer was degraded, indicating that proteolytic activity remained after acid extraction and two forms of chromatography.

Binding and degradation of 125I-gastrin by plasma membranes from homogenized rat gastric mucosa.

Binding of 125I-gastrin to the 270-30,000 g fraction from homogenized rat oxyntic mucosa was studied. 'Specific' binding was calculated by subtracting the binding at excess cold gastrin from the binding with labelled gastrin (250 pM) only. At 30 degrees C specific binding rose rapidly to a short-lived maximum before falling gradually, whereas at 15 degrees C and 0 degree C specific binding rose gradually to a higher plateau level. The reduced binding at 30 degrees C could be caused by degradation of either the tracer or the binding site or by a combination of these two events. Degradation of 125I-gastrin was evaluated by trichloroacetic acid (TCA) precipitation, fast protein liquid chromatography, and binding to a gastrin antibody (immunoreactivity). The effect of incubation on the binding site was evaluated by preincubation of the homogenate fraction before adding gastrin. In separate studies, the proteolytic activity of the homogenate fraction was studied by TCA precipitation of radioactive casein. Different enzyme inhibitors tested were virtually ineffective in preventing gastrin and casein degradation. Only lowering the incubation temperature to 15 degrees C or lower could prevent this degradation. The reduced and transient binding of 125I-gastrin at 30 degrees C most probably reflects tracer degradation. Accordingly, the gastrin binding experiments were performed at 15 degrees C. Only homogenates from the oxyntic area of the stomach bound 125I-gastrin specifically and with a Kd of 0.8 nM (Scatchard analysis). However, micromolar concentrations of unlabelled gastrin were required to inhibit half maximal binding of the tracer. The tracer binding was unaffected by secretin, slightly reduced by a CCK-9 analogue, and more markedly reduced by pentagastrin.

Cyclo(His-Pro), a metabolite of thyrotropin-releasing hormone: Specific binding to rat liver membranes

[ 3H]cyclo(His-Pro) bound with high affinity (59 nM) to a single class of sites in rat liver plasma membranes, without significant tracer degradation during equilibration for 60 min at 0°C. Binding was specific and saturable (3.9 pmol/mg protein), and were increased by the addition of K +, Mg ++ and Na + at optimal concentrations, but not of Ca ++ at all concentrations tested. In vivo administration of cyclo(His-Pro), but not thyrotropin-releasing hormone, to rats caused the downregulation of cyclo(His-Pro)-binding sites with decreases in specific binding numbers but did not change binding affinity.

Uptake and disposal of BSA-coated latex particles by the rat mesangium: reaction with subsequently administered heterologous antiserum.

Mesangial uptake and disposal of antigen-coated latex particles and the ability of subsequently injected antibody to maintain complexed antigen in the rat mesangium has been investigated. Carboxylate-modified latex particles, coated with bovine albumin (BSA) were injected i.v. to 36 Wistar rats. Twenty-two rats (group 1) were not treated further. Fourteen rats (group 2) received rabbit anti-BSA antiserum i.v. and i.p. 24 h later. Control groups were injected with uncoated, unmodified latex particles or soluble BSA with and without subsequent antibody administration. Latex was present in the mesangial matrix of rats in group 1 at 1 h in association with a diffuse mesangial distribution of BSA. At 24 h, BSA staining was markedly reduced and extracellular latex was no longer observed. Intracellular latex aggregates were present in experimental and control groups at 24 h-14 days in cytoplasmic vacuoles of hypertrophic mesangium which showed minor infiltration by macrophage-like cells. Progressive removal of latex aggregates coincided with declining mesangial reactivity. Rapid disappearance of antigen apparently results from local degradation of tracer in the mesangium. Antibody administration preserves BSA in the mesangium due to immune complex formation and is associated with retention of ingested latex by mesangial cells. However, efficient disposal of glomerular immune deposits by the mesangium appears to minimize infiltration by monocytes and prevents aggravation of glomerular inflammation.

The binding of somatostatin to rat synaptosomal membranes: Effects of detergent solubilisation and of bacitracin

Specific high and low affinity binding sites for somatostatin have been demonstrated in rat brain synaptosomal membranes using 125I [Tyr″] somatostatin tracer. When incubations were carried out in the presence of low concentrations of bacitracin (0.01% w/v), a 66% increase in association constant of the high affinity binding site to K a = 1.71 × 10 10 M −1 and a 70% increase in the K a of the low affinity site to 0.034 × 10 10 M −1 were observed; a similar (50%) increase in the binding capacity of the low affinity site but no increase in the binding capacity of the high affinity site was seen. These results may indicate a direct effect of bacitracin on the membrane that is distinct from its effect as an inhibitor of tracer degradation. The somatostatin-binding capacity of the membranes was solubilised by treatment with Triton X-100 and the apparent molecular weight of the receptor-ligand-detergent micelle complex was estimated by gel filtration to be approximately 250,000.

Identification of the angiotensin II receptor in rat mesenteric artery.

Specific binding sites of high affinity and low capacity for 125I-angiotensin II have been identified in a membrane fraction derived from arterial arcades of the rat mesentery. Heterogeneity of binding sites and extensive tracer degradation necessitated the use of nonlinear regression methods for the analysis of radioligand binding data. Forward and reverse rate constants for the high affinity sites obtained by three experimental approaches were in good agreement and gave a dissociation equilibrium constant (Kd) of 19-74 pM (95% confidence interval). Affinities for a number of angiotensin-related peptides calculated from competitive binding curves were in the order 125I-angiotensin II = angiotensin II greater than angiotensin III greater than [Sar1,Ile8]angiotensin II greater than [Sar1,Gly8]angiotensin II. Angiotensin I and biochemically unrelated peptides had virtually no effect on binding of tracer angiotensin II. The divalent cations Mn2+, Mg2+ and Ca2+ stimulated 125I-angiotensin II binding at concentrations of 2-10 mM, as did Na+ at 50-100 mM. In the presence of Na+ or Li+, K+ had a biphasic effect. The chelating agents EDTA and EGTA were inhibitory, as were the thiol reagents dithiothreitol and cysteine. This study defined angiotensin II binding sites in a vascular target tissue of sufficiently high affinity to interact rapidly with plasma angiotensin II at physiological concentrations.

Solubilized adrenal angiotensin II receptors: studies on the site of action of sodium and calcium ions, and on the role of disulfide bridges

The zwitterionic detergent, 3[(3-cholamidopropyl)dimethylammonio)-1 -propane sulfonate (CHAPS), was used to extract angiotensin receptors from the adrenal zona glomerulosa of two species, the rat and beef. The solubilized receptors retained the different properties displayed by particulate receptors in these two species, as well as a preserved affinity and specificity. Moreover, the adrenal receptors lost their ability to be affected by sodium and calcium ions, and by guanyl nucleotides, indicating that the site of action of these modulators is distinct from the receptor itself. Dithiothreitol treatment enhanced the binding of agonist ([ 125I]angiotensin II) and antagonist ([ 125I](Sar 1Ala 8-angiotensin II) to particulate and solubilized rat adrenal receptors, an effect attributable to inhibition of tracer degradation. In contrast, agonist and antagonist binding to particulate bovine receptors was decreased by dithiothreitol, but agonist binding to soluble receptors was increased. In addition to showing important species differences in angiotensin-degrading activity of adrenal receptor preparations, this work suggests that bovine receptors may contain essential sulfhydryl groups for binding and recognition of angiotensin peptides as agonists or antagonists.

Evidence for separate handling in vivo of different regions of the insulin molecule using A14- and B1-labeled insulin tracers.

To compare the metabolic characteristics and degradation of insulin tracers labeled unselectively, selectively at the A14 position (A14-monoiodoinsulin), and selectively at the B1 position (B1-monoiodoinsulin), we have followed the time course of disappearance of intact (immunoprecipitable [IP] and trichloroacetic acid [TCA] precipitable) iodoinsulin after bolus injection into greyhounds. We have used noncompartmental analysis to determine metabolic clearance rate (MCR) and apparent distribution space (DS). We have also measured the appearance of non-IP- and non-TCA-precipitable fragments, and have developed a mathematical model using compartmental analysis to explain the observed differences. B1-Monoiodoinsulin has a significantly higher MCR (16.3 ml/min/kg) than both A14-monoiodoinsulin (10.6 ml/min/kg) and unfractionated tracers (7.6 ml/min/kg) as determined by immunoprecipitation, and reaches the values observed for native insulin in greyhounds. MCR values obtained by TCA precipitation are approximately one-half of those obtained by IP for all 3 tracers. The concentration of non-IP fragments is significantly lower with B1-monoiodoinsulin than with the other tracers. Compartmental analysis suggests this to be due to greater intracellular retention of the B1 moiety during the experimental period. We conclude that: (1) by the criterion of MCR, B1-monoiodoinsulin seems to behave more like native insulin than other preparations tested; (2) the reduced MCR of A14-monoiodoinsulin raises doubts about its validity as a tracer for insulin; (3) a high-molecular-weight product of insulin degradation, which includes both the B1 and the A14-A19 regions of the molecule, is released into the circulation; and (4) smaller fragments containing A14-A19 reappear in the circulation more rapidly than fragments containing B1.(ABSTRACT TRUNCATED AT 250 WORDS)

Diffusion of macromolecules in hyaluronate gels. I. Development of methods and preliminary results.

A technique for measuring the diffusion coefficients of high molecular weight solutes in biological polymers, which requires only small quantities of tracer and substrate is described. The influences on the distribution of the tracer of non-ideal injections, of tracer contamination and degradation, and of various interactions between tracer and substrate are analysed theoretically in order that they may be recognised, and in some cases corrected for, in the experimental data. The experimental technique is tested by measurements of the diffusion coefficient of albumin in hyaluronate gels of 0.5%-2.5% (w/v) concentrations and preliminary data are presented on the diffusion of rabbit lipoproteins in similar gels.

‘Dynorphin’ in plasma: enzymatic artifact and authentic immunoreactivity

The potent opioid peptide dynorphin (DYN) is found in posterior pituitary vasopressinergic neurones and in adrenal medullary cells suggesting that secretion into plasma is likely. We have developed a sensitive radioimmunoassay in order to study plasma DYN in man. It transpired that extraction prior to assay was essential since unextracted plasma caused gross and non-parallel inhibition of binding of tracer. Plasma extracted using leached silica glass (Vycor) caused inhibition of tracer binding which diluted in parallel to synthetic DYN suggesting the presence of substantial amounts of DYN-like immunoreactivity (irDYN) in plasma. Further investigation however demonstrated that this irDYN was artifactual and caused by enzymatic degradation of tracer. Although use of Seppak C18 cartridges resulted in reliable extraction of synthetic procine DYN from acidified plasma, we have not detected irDYN in any plasma so far studied using this technique. However, extraction of non-acidified plasma using our antibody coupled to Sepharose CNBr-activated 4B followed by gel filtration chromatography demonstrated a single peak of irDYN of molecular size similar to DYN. These data suggested that a small amount of a DYN-like peptide does circulate in human plasma although this is not identical to porcine DYN(1–17). The implication of our results for the measurement of other similar peptides in plasma is discussed.

Salivary gland glucagon is a fictitious substance due to tracer-degrading activity resistant to protease inhibitors

A high level of glucagon immunoreactivity was apparently detected in acid-saline extract from rat submandibular glands, but tracer glucagon added to the assay mixture was mostly damaged in spite of the presence of protease inhibitors commonly used in radioimmunoassay. Gel-filtration of the extract on a Bio-Gel P-10 column revealed strong tracer-degrading activity at the void fraction where the apparent immunoreactivity was eluted. Serial changes in apparent immunoreactivity of the extract fit well on the theoretical curve of an exponential tracer degradation. These findings indicate that the salivary gland glucagon is a fictitious substance due to tracer degradation during radioimmunoassay. Further study revealed that the glucagon molecule was hydrolyzed at the arginyl bonds and split into two fragments during incubation with the acid-saline extract from rat submandibular glands.

“Immunoreactive dynorphin” in escherichia coli: Tracer degradation by a heat-stable endopeptidase

Escherichia coli cells contain and secrete into the growth medium a heat-stable tryptic enzyme, which degrades the radiolabeled dynorphin tracer peptide during radioimmunoassay, simulating the presence of “immunoreactive dynorphin”. The validity of an immunoassay procedure, in each new application, must remain in doubt until tracer integrity has been proved.

Characterization of somatostatin receptors in the rat adrenal glomerulosa zone.

Specific receptors for somatostatin have been identified and characterized in the rat adrenal glomerulosa zone in vivo and in vitro by binding studies with [125I]iodo-Tyr-somatostatin. In adult rats, the injection of [125I]iodo-Tyr-somatostatin was followed by rapid uptake of the labeled peptide in several tissues. The highest uptake was in the adrenal capsule, with a tissue to blood ratio of 4.5, followed by kidney, anterior pituitary, and liver with tissue to blood ratios of 2.0, 1.8, and 1.4, respectively. In vitro binding studies with adrenal capsular particles were performed at 16 C in the presence of bacitracin and thimerosal to reduce tracer degradation. Under these conditions, binding of [125I]iodo-Tyr-somatostatin to capsular membrane-rich fractions reached a steady state within 30-40 min and remained at a plateau for 120 min. Dissociation of bound somatostatin from its adrenal receptors was also rapid, with an initial half-time of 5 min. Equilibrium binding of somatostatin to adrenal capsular particles was saturable and of high affinity, with an association constant (Ka) of 1.5 x 10(10) M-1. The receptor-binding activities of several somatostatin analogs were consistent with their potencies as inhibitors of angiotensin II-stimulated aldosterone production in adrenal capsular cells and with their known biological activities upon GH release. These findings demonstrate that high affinity receptors with structural and biological specificities for somatostatin are present in the adrenal glomerulosa zone. Such receptors serve as the regulatory sites through which somatostatin inhibits the action of angiotensin II upon aldosterone production in the adrenal glomerulosa cell.

The nature of big plasma somatostatin: Implications for the measurement of somatostatin-like immunoreactivity in human plasma

Big plasma somatostatin (BPS) represents an artifact of measurement. High-molecular-weight globulins (α, β, and γ) in human plasma inhibit, in a concentration-dependent manner, the binding of radiolabeled somatostatin analogs to antibody directed against somatostatin. The magnitude of inhibition varies with antibody and plasma sample and is greatest for the α-globulin fraction. The mechanism of inhibition involves binding of plasma globulins to antibody, thereby blocking tracer-binding sites, and does not involve inhibition by somatostatin bound noncovalently to plasma proteins or tracer degradation. Thus BPS arises from a property of plasma rather than of somatostatin and so it is suggested that this mechanism may account for the presence of other “big” forms of hormones in plasma.

Receptor binding and processing of epidermal growth factor by human breast cancer cells.

Epidermal growth factor (EGF) stimulates the growth of human breast cancer cells (MCF-7). To better define the mechanisms for these effects, we studied receptor binding and subsequent processing of [l25I]EGF in cells growing in monolayer culture under conditions in which an effect on growth is observed. Binding was temperature dependent and was higher at reduced temperature. At 37 C, binding rapidly reached a maximum (20–60 min) and then gradually decreased. Binding was slower at a reduced temperature. High affinity was demonstrated by the ability of unlabeled EGF to compete for [125I]EGF binding; hah7 maximal displacement was observed at an EGF concentration that also resulted in half-maximal biological activity (1–2 ng/ml). Scatchard plots derived from these data or from those of studies using increasing concentrations of [125I]EGF were curilinear and superimposable. Plots from binding data obtained at 0 C were also curvilinear, suggesting that the processing and degradation of tracer alone cannot expla...

Development and validation of a radioimmunoassay for beta-endorphin-related peptides.

A radioimmunoassay method suitable for measuring levels of B-endorphin, B-lipotropin and proopiocortin in tissue and plasma extracts was developed and the method was evaluated by using 3 independently prepared antisera. Of the several variables tested the choice of assay buffer and test tubes and the purification of tracer were found to be the most critical in the successful performance of B-endorphin radioimmunoassay. The prevention of degradation of tracer during incubation was also necessary when crude tissue extracts or plasma were assay. The sensitivities of the assays obtained with the 3 antisera utilized (Bendo 2, K2 and RB 100) were 1, 2.8 and 4 fmol B-endorphin per tube. All the antisera crossreacted equimolarly with B-lipotropin. The method was employed to measure the levels of B-endorphin immunoreactivity in rat pituitary and plasma by separating the different immunoreactants by gel filtration. It was found that both pituitary and plasma contain significant amounts of proopiocortin, B-lipotropin and B-endorphin, the molar proportions being 10:33:57 in pituitary and 15:15:17 in plasma, respectively. Both anterior and posterior lobes of rat pituitary were found to contain all the three immunoreactants. However, anterior lobe contained mostly the larger molecules, while posterior lobe was rich in B-endorphin. No absolute levels of the immunoreactants could be obtained due to the heterologous system used. Moreover the elution pattern of the immunoreactivity was found to be dependent on the conditions used for elution in gel filtration: higher proportion of the immunoreactivity eluted like B-endorphin when the elution was done in dissociating conditions (6 M urea) compared to elution with ordinary buffers.

Regulation of pituitary receptors for gonadotropin-releasing hormone during the rat estrous cycle.

A radioiodinated superagonist analog of gonadotropin-releasing hormone (GnRH:[D-Ser(TBu)6]des-GLy10-GnRH N-ethylamide) was used to quantitate the GnRH receptor content of single pituitary glands. This ligand binds with high affinity (Ka = 4.9 X 10(9) M-1) to a single class of sites in pituitary homogenates, without significant tracer degradation, during equilibration for 80 min at 4 C. The GnRH receptor content of adult male rat pituitaries [112 +/- 6.4 (SE) fmol/gland; n = 9] was similar to that of adult female rat pituitaries obtained at estrus or metestrus (104.8 +/- 6.4 fmol/gland, n = 13). Between 1800 h on metestrus and 1800 h on diestrus, the pituitary content of GnRH receptors increased to 200 fmol/gland or greater in each of three separate experiments. The pituitary content of GnRH receptors remained elevated until the time of the serum LH surge, then consistently fell sharply to metesterus levels. These data imply a physiological relevance for the GgRH-binding sites measured by radioassay and demonstrate their relationship to the events of the estrous cycle. The increased GnRH receptor content from the evening of diestrus until late in the afternoon of proestrus may contribute to the enhanced sensitivity of the pituitary to GnRH during proestrus. The close temporal relationship between rising blood estrogen levels and increasing pituitary GnRH receptors suggests that steroid-mediated receptor induction occurs before proestrus. Such an effect of estrogen could be exerted directly upon the pituitary gland or indirectly via the hypothalamus to increase the release of endogenous GnRH, which subsequently induces its own receptors in pituitary gonadotrophs.