β-Adrenergic receptor activity of cerebral microvessels is reduced in aged rats

The effect of age on beta-() adrenergic receptor number (B_max) and adenylate cyclase (AC) activity was determined in microvessels isolated from male F-344 rats at 3, 18, and 24 months of age. Scatchard analysis of [¹²⁵I]iodocyanopindolol (ICYP) binding indicated reduced Bmax (fmol/mg) of microvessels isolated from 24 month old rats (27.2±4.9) compared with 3 month old (50.4±5.2) and 18 month old rats (p<0.01) (61.4±7.6). The basal AC activity (pmol cAMP/mg) in 24 month old rats (32.0 ±6.7) and in 18 month old rats (30.4±2.1) were significantly reduced compared to the basal activity in the young (50.1±4.2). The net isoproterenol or NaF stimulated AC activity in 24 month old rats (zero and 15.6±8.5 respectively) was also reduced compared to young rats (10.1±3.9 and 166.0±21.2 respectively). It is concluded that aging is associated with reduced isoproterenol stimulated AC activity of cerebral microvessels. This reduction is the product of reduced -adrenergic receptor number and reduced activity of AC in aged rat cerebral microvessels.     

High affinity binding of reovirus type 3 to cells that lack beta adrenergic receptor activity

A previous report demonstrated both immunological crossreactivity and structural similarity between the mammalian beta adrenergic receptor and the cell surface receptor for the reovirus type 3 (14). We now demonstrate that reovirus type 3 can bind selectively and with high affinity to cells that lack beta adrenergic receptor activity (L-cells). The present study was also designed to determine what effect reovirus binding has on beta adrenergic receptor function in cells (DDT1) that possess an intact ligand binding site. Based on computer analysis of reovirus competitive inhibition curves, the apparent dissociation binding constants (Kd) for reovirus binding to DDT1 and L-cells are 0.1 nM and 0.25 nM, respectively. High affinity [125I]-iodocyanopindolol (CYP) binding to beta adrenergic receptors can also be demonstrated in DDT1 cells but not in L-cells. In agreement with these ligand binding studies, adenylate cyclase activity is stimulated by the beta receptor agonist isoproterenol in DDT1 cell membranes but not in L-cell membranes. In addition, isoproterenol increases cAMP levels in DDT1 cells but not in L-cells. Neither reovirus serotype stimulates cAMP levels in either cell line, nor do they influence beta-adrenergic agonist stimulation of cAMP in DDT1 cells. These results argue against identity of the receptors for reovirus type 3 and beta adrenergic ligands.     

β2-Adrenoceptor Agonist-Induced Down-Regulation After Short-Term Exposure

Abstract

We examined the effect of duration of β₂-adrenergic receptor (β₂AR) occupancy by isoproterenol on specific binding of ¹²⁵l-lodocyanopindolol (¹²⁵I-ICYP) in membranes from rat L6 myoblasts. Ten minute exposure caused a time- and concentration-dependent maximal decrease in ¹²⁵-?YP binding 24 hours after exposure equal to that following continuous exposure (p < 0.05). Low temperature, concanavalin A, H89 and ICI 118,551 blocked the decline in ¹²⁵I-ICYP binding during the first hour following exposure probably representing receptor sequestration to a compartment or change to a form incapable of ligand binding. Compared to controls, receptor binding 4 and 24 hours following exposure was reduced 56 ± 8.7% and 72 ± 8.8%, respectively (p < 0.05), and was blocked by ICI 118,551 but not CGP12177. Isoproterenol-induced, but not forskolinstimulated, cAMP accumulation was reduced 35% 24 hours following exposure (p < 0.05). ¹²⁵I-ICYP binding in intact L6 cells 4 and 24 hours after exposure were respectively 56 ± 8.9 and 61 ± 13% of controls (p < 0.05). Following agonist exposure, CHO cell membranes expressing human β₂ARs exhibited ¹²⁵I-ICYP binding 85 ± 2.0% and 6 ± 2.8% of control values 4 and 24 hours, respectively (p < 0.05). A model predicting that full occupation of the β₂AR activates receptor degradation explains our results that agonist-induced down-regulation of β₂AR does not require continuous presence of the agonist.     

Characterization of functional calcitonin gene-related peptide receptors on rat lymphocytes.

Calcitonin gene-related peptide (CGRP), a vasoactive neuropeptide present in peripheral neurons, is released at local sites of inflammation. In these studies specific high affinity adenylyl cyclase linked CGRP receptors were characterized on rat lymphocytes. The distribution, affinity, and specificity of CGRP receptors was analyzed by radioligand binding. 125I-[His10]CGRP binding to rat lymphocytes was rapid, reaching equilibrium by 20 to 30 min at 22 degrees C, and dependent on cell concentration. The dissociation constants, Kd, for the CGRP receptor on purified T and B lymphocytes are 0.807 +/- 0.168 nM and 0.387 +/- 0.072 nM and the densities are 774 +/- 387 and 747 +/- 244 binding sites/cell, respectively. Competition binding studies determined that rat CGRP inhibits 125I-[His10]CGRP binding to lymphocytes with the highest affinity (Ki = 0.192 +/- 0.073) followed by human CGRP and the CGRP receptor antagonist CGRP8-37. 125I-[His10]CGRP binding to rat lymphocytes was not inhibited by the neuropeptides substance P, calcitonin, or neuropeptide Y. Lymphocyte CGRP receptor proteins were identified by affinity labeling by using disuccinimidyl suberate to covalently cross-link 125I-[His10]CGRP to its receptor. Specifically labeled CGRP binding proteins visualized by SDS-PAGE analysis had molecular masses of 74.5 and 220 kDa. A third high molecular mass protein band which did not penetrate the gel was also observed. In functional studies, CGRP stimulated a rapid, sustained increase in cAMP with an ED50 of approximately 8 pM. In experiments comparing optimal concentrations of isoproterenol, a beta 2-adrenergic agonist, and CGRP, intracellular cAMP elevation after isoproterenol treatment returned to basal levels by 30 min, whereas cAMP was still elevated at 60 min after CGRP treatment. The response to CGRP was specific in that it could be completely blocked by CGRP8-37. The presence of high affinity functional CGRP receptors on T and B lymphocytes provides evidence for a modulatory role for CGRP in regulating lymphocyte function.     

Beta 2-adrenoceptor agonist-induced down-regulation after short-term exposure.

We examined the effect of duration of beta 2-adrenergic receptor (beta 2AR) occupancy by isoproterenol on specific binding of 125I-lodocyanopindolol (125I-ICYP) in membranes from rat L6 myoblasts. Ten minute exposure caused a time-and concentration-dependent maximal decrease in 125I-ICYP binding 24 hours after exposure equal to that following continuous exposure (p < 0.05). Low temperature, concanavalin A, H89 and ICl 118,551 blocked the decline in 125I-ICYP binding during the first hour following exposure probably representing receptor sequestration to a compartment or change to a form incapable of ligand binding. Compared to controls, receptor binding 4 and 24 hours following exposure was reduced 56 +/- 8.7% and 72 +/- 8.8%, respectively (p < 0.05), and was blocked by ICl 118,551 but not CGP12177. Isoproterenol-induced, but not forskolin-stimulated, cAMP accumulation was reduced 35% 24 hours following exposure (p < 0.05). 125I-ICYP binding in intact L6 cells 4 and 24 hours after exposure were respectively 56 +/- 8.9 and 61 +/- 13% of controls (p < 0.05). Following agonist exposure, CHO cell membranes expressing human beta 2ARs exhibited 125I-ICYP binding 85 +/- 2.0% and 6 +/- 2.8% of control values 4 and 24 hours, respectively (p < 0.05). A model predicting that full occupation of the beta 2AR activates receptor degradation explains our results that agonist-induced down-regulation of beta 2AR does not require continuous presence of the agonist.     

Anesthetic Barbiturates Enhance Gsα-Dependent Cyclic AMP Production in S49 Mouse Lymphoma Cells

Abstract: Cyclic AMP (cAMP) regulates many important physiological processes. Barbiturates influence cAMP regulation, possibly through effects on G proteins. This study used intact S49 mouse lymphoma cells to characterize the role of G proteins in the effect of barbiturates on cAMP regulation. cAMP accumulation was determined in intact S49 WT (wild-type) and S49 cyc^? cells (the G_sα-deficient mutant) by measuring the conversion of [³H]-ATP to [³H]cAMP in cells preloaded with [³H]adenine. Pentobarbital enhanced cAMP accumulation in WT cells in the absence (basal) or presence of isoproterenol but had no effect on the EC₅₀ for isoproterenol. This effect was dose dependent with a 50–60% enhancement at 2 mM pentobarbital. Pentobarbital did not affect forskolin-stimulated cAMP accumulation in WT cells. In cyc^? cells, basal and forskolin-stimulated cAMP accumulation were stimulated only at the highest concentration of pentobarbital used (2 mM). Pentobarbital did not affect the inhibition of cAMP accumulation by somatostatin in WT cells, and pertussis toxin treatment of WT cells did not affect the action of pentobarbital on cAMP accumulation. Pentobarbital did not affect isoproterenol-stimulated adenylyl cyclase activity in whole-cell homogenates or membranes prepared from WT cells. The S-(?)-isomer of pentobarbital enhanced isoproterenol-stimulated cAMP accumulation more than the R-(+)-isomer. Phenobarbital and barbituric acid did not enhance isoproterenol-stimulated cAMP accumulation, whereas the anesthetic barbiturates hexobarbital, pentobarbital, and thiopental all enhanced activity. These results suggest that pentobarbital enhances cAMP accumulation in intact WT cells by a mechanism that is dependent on G_sα but independent of G_i. The properties of barbiturates that are responsible for the enhancement of cAMP accumulation may be related to the properties that are responsible for producing sedation and anesthesia.     

Production of antibodies that inhibit the binding of insulin to its receptor

In this study, we report a procedure for producing antisera that block the binding of ¹²⁵I-insulin to its receptor. After 2 injections with intact IM-9 cultured human lymphocytes, the antisera from 8 of 17 $\text{Balb}\text{C}$ mice inhibited the binding of ¹²⁵I-insulin to its receptor on IM-9 cells by 50% or greater. One antiserum at dilutions of 1:200 and 1:50 inhibited the binding of ¹²⁵I-insulin by 50% and 80%, respectively. Four lines of evidence indicated that the inhibition of ¹²⁵I-insulin binding by this antiserum was due to a specific immunoglobulin directed against the insulin receptor. First, removal of the immunoglobulin fraction of the antiserum resulted in a complete loss of its inhibitory activity. Second, the antiserum inhibited the binding of ¹²⁵I-insulin to its receptor on both human cultured lymphocytes and human placenta particles. Third, the antisera bound solubilized insulin-receptor complexes. Finally, the antiserum did not inhibit the binding of ¹²⁵I-human growth hormone to its receptor on IM-9 lymphocytes. These studies demonstrate therefore, a simple method for producing antibodies that block the binding of ¹²⁵I-insulin to the human insulin receptor.     

Hormone action at the membrane level VI. Binding of (—)-[3H]dihydroalprenolol and (±)-[3H]isoproterenol to turkey erythrocytes and correlation with adenylate cyclase activity

The binding of (±)-[³H]isoproterenol and (—)-[³H]dihydroalprenolol to intact turkey erythrocytes was studied using a rapid centrifugation technique. The binding of both ligands is rapid, dissociable, stereospecific and inhibited by (—)-propranolol. The total number of isoproterenol binding sites is 2800 sites/ cell. This consists of a low and high affinity site both of which show stereospecific binding. The high affinity isoproterenol site has a K_d of 15.5—19.5 nM and has 600 sites/cell. The low affinity isoproterenol site has a K_d of 195 nM and has 2200 sites/cell. The binding of (—)-[³H]dihydroalprenolol shows one type of site with a K_d of 7.8 nM and has 2500 sites/cell. The agonists epinephrine, norepinephrine, soterenol and p-hydroxyphenylisoproterenol which were tested by competition for binding showed a 6—25-fold greater affinity for the high affinity site determined by (±)-[³H]isoproterenol as compared to the (—)-[³H]dihydroalprenolol binding site. However, the antagonists propranolol, practolol and metrapolol showed similar affinities for the binding sites as determined by competition of binding of either labeled isoproterenol or dihydroalprenolol. These studies indicate that isoproterenol binding can recognize two independent stereospecific β-adrenergic receptors or can recognize two different conformational states of a single receptor. Provisional calculations are made on the turnover number of adenylate cyclase under physiological conditions using intact erythrocytes. The turnover number is 4000 molecules of cyclic AMP/10 min per high affinity receptor.     

Phospholipid methyltransferase activity in diabetic rat fat cells: effect of isoproterenol and insulin

The effects of isoproterenol and insulin on phospholipid methyltransferase (PLMT) activity were investigated in adipocytes from control and streptozotocin-diabetic rats. PLMT activity was assayed by measuring the rate of incorporation of ³H-methyl groups from S-adenosyl-L-[methyl-³H] methionine into phospholipids. Basal PLMT activity was higher in adipocytes from diabetic animals. Treatment of adipocytes with isoproterenol induced a concentration-dependent stimulation of PLMT activity. In control adipocytes, the maximal effect was obtained at 100 nM isoproterenol with 2.3 fold increase in PLMT activity and a half maximal effect at 25 nM. In adipocytes from diabetic rats, a lower dose of isoproterenol (10 nM), caused 1.2 fold increase with a half maximal effect at 4 nM. Addition of 100 nM insulin inhibited basal PLMT activity and the stimulatory effect of isoproterenol in both types of adipocytes. The -adrenergic blocking agent propranolol inhibited the stimulatory effect of isoproterenol on PLMT activity in control and diabetic adipocytes. Intracellular concentration of cAMP was higher in diabetic adipocytes but decreased to normal values after incubation in the presence of insulin.     

Interaction of alpha and beta adrenergic stimulation on aortic ornithine decarboxylase activity

The interaction between beta and alpha adrenergic agonists on regulation of cockerel aortic ornithine decarboxylase (ODC) activity was examined. The beta adrenergic agonist isoproterenol both reduced basal aortic ODC activity and prevented induction of the decarboxylase by the alpha adrenergic agonist methoxamine. 3-Isobutyl-1- methylxanthine (IBMX) similarly reduced basal ODC activity and blocked induction of the enzyme by methoxamine. When given ten minutes before or after methoxamine, isoproterenol prevented aortic ODC induction, but not large sustained increases in blood pressure evoked by the alpha adrenergic agonist. In contrast, when injected three hours after methoxamine, isoproterenol had no effect on already elevated levels of enzyme activity. Addition of isoproterenol (10(-7)M), IBMX (1 mM) or dibutyryl cAMP (2.5 mM) to isolated aortic segments cultured in minimal salts-glucose media evoked decreases in basal levels of ODC activity resembling those observed in the intact animal. These results suggest that the balance between alpha and beta adrenergic stimulation may be an important feature of the regulation of polyamine biosynthesis in artery wall cells.     

Loss of adenylate cyclase hormonal sensitivity in chemically transformed epithelial cells.

The hormonal sensitivity of adenylate cyclase from a normal rat liver epithelial cell line (K16) and its chemically transformed derivative (W8) were compared. Intact normal rat liver cells had markedly increased cAMP levels after brief exposure to epinephrine, isoproterenol, norepinephrine or prostaglandin E₁. In contrast, the cAMP levels of chemically transformed cells were relatively unaffected by these same compounds even after prolonged incubation. A comparison of broken cell adenylate cyclase activities revealed a decreased basal activity in the chemically transformed cells; the response to NaF was similar in the two cell lines, while the response to catecholamines and prostaglandins paralleled the intact cell studies. These data suggest that one reason for loss of adenylate cyclase hormonal responsiveness in chemically transformed rat liver epithelial cells may be a dysfunction or loss of hormone binding sites.     

Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 1: an ovarian stage dependent involvement

The finding that molt-inhibiting hormone (MIH) regulates vitellogenesis in the hepatopancreas of mature Callinectes sapidus females, raised the need for the characterization of its mode of action. Using classical radioligand binding assays, we located specific, saturable, and non-cooperative binding sites for MIH in the Y-organs of juveniles (J-YO) and in the hepatopancreas of vitellogenic adult females. MIH binding to the hepatopancreas membranes had an affinity 77 times lower than that of juvenile YO membranes (K_D values: 3.22 × 10^-8 and 4.19 × 10^-10 M/mg protein, respectively). The number of maximum binding sites (B_MAX) was approximately two times higher in the hepatopancreas than in the YO (B_MAX values: 9.24 × 10^-9 and 4.8 × 10^-9 M/mg protein, respectively). Furthermore, MIH binding site number in the hepatopancreas was dependent on ovarian stage and was twice as high at stage 3 than at stages 2 and 1. SDS-PAGE separation of [¹²⁵I] MIH or [¹²⁵I] crustacean hyperglycemic hormone (CHH) crosslinked to the specific binding sites in the membranes of the J-YO and hepatopancreas suggests a molecular weight of ~51 kDa for a MIH receptor in both tissues and a molecular weight of ~61 kDa for a CHH receptor in the hepatopancreas. The use of an in vitro incubation of hepatopancreas fragments suggests that MIH probably utilizes cAMP as a second messenger in this tissue, as cAMP levels increased in response to MIH. Additionally, 8-Bromo-cAMP mimicked the effects of MIH on vitellogenin (VtG) mRNA and heterogeneous nuclear (hn) VtG RNA levels. The results imply that the functions of MIH in the regulation of molt and vitellogenesis are mediated through tissue specific receptors with different kinetics and signal transduction. MIH ability to regulate vitellogenesis is associated with the appearance of MIH specific membrane binding sites in the hepatopancreas upon pubertal/final molt.     

Effect of Synthetic β-Endorphin-Like Peptide Immunorphin on Human T Lymphocytes

-Endorphin and the synthetic -endorphin-like decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (referred to as immunorphin), corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain, were shown to stimulate concanavalin A-induced proliferation of T lymphocytes from the blood of healthy donors. [Met⁵]Enkephalin and the antagonist of opioid receptors naloxone examined in parallel were inactive. The stimulating effect of -endorphin and immunorphin on T lymphocyte proliferation is not inhibited by naloxone. Studies on receptor binding of ¹²⁵I-labeled immunorphin to T lymphocytes revealed that it binds with high affinity to naloxone-insensitive receptors (K _d = 7.0 ± 0.3 nM)). Unlabeled immunorphin completely inhibits ¹²⁵I-labeled -endorphin specific binding to naloxone insensitive receptors on T lymphocytes (K _i = 0.6 ± 0.1 nM)). Thus, -endorphin and immunorphin interact with common naloxone insensitive receptors on T lymphocytes.     

beta-Adrenergic-responsive activation of extracellular signal-regulated protein kinases in salivary cells: role of epidermal growth factor receptor and cAMP

The -adrenergic receptor agonist isoproterenol exerts growth-promoting effects on salivary glands. In this study, activation of ERKs, members of the mitogen-activated protein kinase family, by isoproterenol was examined in a human salivary gland cell line (HSY). Immunoblot analysis indicated that isoproterenol (10^–5 M) induced transient activation of ERK1/2 (4.4-fold relative to basal at 10 min) similar to that caused by EGF (6.7 fold). Isoproterenol, like EGF, also induced phosphorylation of the EGF receptor. However, inhibition of EGF receptor phosphorylation by the tyrphostin AG-1478 only partially attenuated isoproterenol-induced ERK phosphorylation, whereas EGF-responsive ERK activation was completely blocked. The G_i inhibitor pertussis toxin also caused partial inhibition of isoproterenol-stimulated ERK activation. The cAMP analog 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and the cAMP-elevating agents IBMX and cholera toxin produced transient ERK1/2 activation, similar to the effect of isoproterenol, in HSY cells. The stimulatory effects of isoproterenol and cAMP on ERK phosphorylation were not reduced by the PKA inhibitor H-89, whereas the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidase (PP2) and transfection of a dominant-negative Src construct diminished isoproterenol-induced ERK activation. Isoproterenol induced marked overexpression of the cell growth-related adhesion molecule CD44, and this effect of isoproterenol was abolished by the ERK pathway inhibitor PD-98059. In summary, we show a dual mechanism of isoproterenol-induced ERK phosphorylation in HSY cells—one pathway mediated by EGF receptor transactivation and the other by an EGF receptor-independent pathway possibly mediated by cAMP. Our results also suggest that isoproterenol-induced growth of salivary tissue may involve ERK-mediated CD44 expression. mitogen-activated protein kinase; CD44     

Mutation of alanine 623 in the third cytoplasmic loop of the rat thyrotropin (TSH) receptor results in a loss in the phosphoinositide but not cAMP signal induced by TSH and receptor autoantibodies.

Thyrotropin (TSH) and IgG preparations from patients with Graves' disease increase inositol phosphate as well as cAMP formation in Cos-7 cells transfected with rat TSH receptor cDNA. Mutation of alanine 623 in the carboxyl end of the third cytoplasmic loop of the TSH receptor, to lysine or glutamic acid, results in the loss of TSH- and Graves' IgG-stimulated inositol phosphate formation but not in stimulated cAMP formation. There is no effect of the mutations on basal or P2-purinergic receptor-mediated inositol phosphate formation. The mutations do not affect transfection efficiency or the synthesis, processing, or membrane integration of the receptor, as evidenced by the unchanged amount and composition of the TSH receptor forms on Western blots of membranes from transfected cells. The mutations increase the affinity of the TSH receptor for [125I]TSH and decrease Bmax; however, cells with an equivalently decreased Bmax as a result of transfection with lower levels of wild type receptor do not lose either TSH-induced inositol phosphate formation or cAMP signaling activity. Thus, in addition to discriminating between ligand-induced phosphatidylinositol bisphosphate and cAMP signals, the mutation appears to cause an altered receptor conformation which affects ligand binding to its large extracellular domain.     

Differential effects of paraoxon on the M3 muscarinic receptor and its effector system in rat submaxillary gland cells

The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M₃ subtype of the muscarinic receptor and receptor-coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [³H]quinuclidinyl benzilate ([³H]QNB) was most sensitive to atropine and the M₃-specific antagonist 4-DAMP followed by pirenzepine and least sensitive to the cardioselective M₂ antagonist AFDX116. This, and the binding characteristics of [³H]4-DAMP, confirmed that the muscarinic receptors in this preparation are of the M₃ subtype. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems. Paraoxon (100 μM) reduced B_max of [³H]4-DAMP binding from 27 ± 4 to 13 ± 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolininduced cAMP formation in SMG cells with an EC₅₀ of 200 nM, but paraoxon was > 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 μM atropine, that by paraoxon was unaffected by up to 100 μM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect β-adrenoreceptor function in the preparation, since it did not affect the 10 μM isoproterenol-induced cAMP synthesis, which was inhibited totally by 10 μM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC-stimulated PI metabolism in the SMG cells. It is suggested that paraoxon binds to two different sites in these SMG cells. One is an allosteric site on the M₃ muscarinic receptor which affects ligand binding and may modulate receptor function. The other site may be on the G_i proteinadenylyl cyclase system, and produces CBC-like action, that is, inhibition of the forskolin-stimulated [³H]cAMP synthesis, and is unaffected by atropine inhibition of the muscarinic receptor. This adds to the complexity of paraoxon actions on muscarinic receptors and their effector systems.     

Reactivity of bacterial Fc receptors with bovine immunoglobulins: implications and limitations for quantitative immunochemistry

Summary The interaction of bovine immunoglobulins with staphylococcal Protein A and a group C streptococcal bacterial Fc receptor (FcRc) were compared. The isolated group C streptococcal receptor was reactive with both bovine IgG₁ and IgG₂. The reactivity of the streptococcal FcRc with IgG₂ was approximately 40 fold greater than that observed with IgG₁. By contrast, protein A reacted only poorly with bovine IgG₂ and no detectable reactivity was observed with IgG₁. A two stage competitive binding assay to measure bovine IgG in serum and secretions using ¹²⁵I-labeled protein A as tracer was developed. This assay was found to be sensitive and reproducible and was used to measure serum IgG levels in cattle of differing ages and breeds.     

Amyloid Beta Peptide 1-40 and the Function of Rat Hippocampal Hemicholinium-3 Sensitive Choline Carriers: Effects of a Proteolytic Degradation In Vitro

Effects of amyloid beta peptide 1-40 (Abeta) and of plant cysteine proteases bromelain and papain on the high-affinity uptake of choline (HACU) and the specific binding of [³H]hemicholinium-3 ([³H]HC-3) have been investigated on hippocampal synaptosomes from young adult male Wistar rats under basal and stimulated conditions (55 mM KCl). Depolarization increased significantly the HACU levels (the changes were predominantly in Vmax) and mildly the [³H]HC-3 binding (the changes especially in K_D). Nonaggregated Abeta at low nM concentrations suppressed the depolarization effects but was ineffective under basal conditions during a short-term incubation. Higher M concentrations decreased the HACU and binding under basal conditions in a time-dependent manner. The binding changes were firstly associated with alterations in K_D and secondarily were accompanied also by a drop in Bmax. The results suggest that Abeta directly influences high-affinity carriers, inhibits their transport activity and enhances their sensitivity to proteolytic cleavage. Stimulation increases the sensitivity of carriers to the interaction with Abeta.     

Role of Ca2+-calmodulin dependent phospholamban phosphorylation on the relaxant effect of β-adrenergic agonists

The role of the Ca²⁺-calmodulin dependent pathway of phospholamban phosphorylation on the relaxant effect of -adrenergic agonists was studied in isolated perfused rat heart. Administration of the calmodulin antagonist W7 or lowering [Ca]₀ from 1.35 mM (control) to 0.25 mM, were used as experimental tools to inhibit the Ca²⁺-calmodulin dependent protein kinase activity. 3×10^–8 M isoproterenol increased cAMP levels from 0.613±0.109 pmol/mg wet weight to 1.581±0.123, phospholamban phosphorylation from 36±6 pmol³²P/mg protein to 277±26 and decreased time to half relaxation (t1/2) from 61±2 msec to 39±2. Simultaneous perfusion of isoproterenol with 10^–6 M W7, decreased phospholamban phosphorylation to 170±23 and prolongated t1/2 to 47±3 but did not affect the increase either in cAMP levels or myocardial contractility produced by isoproterenol. Similar effects on phospholamban phosphorylation and myocardial relaxation were obtained when isoproterenol was perfused in low [Ca]₀. Low [Ca]₀ did not affect the increase in cAMP elicited by isoproterenol but offset the positive inotropic effect of the -agonist.The results suggest a physiological role of the Ca²⁺-calmodulin dependent phospholamban phosphorylation pathway as a mechanism that supports, in part, the -adrenergic cardiac relaxant effect.