Effects of IL-1 and cortisol on beta-adrenergic receptors, cell proliferation, and differentiation in cultured human A549 lung tumor cells

The effects of IL-1 and cortisol, and their interactions on the density of beta-adrenergic receptors (beta AR), cell proliferation, and the adherence of cells to plastic were studied in cultured human A549 lung tumor cells. The density of beta AR, assayed by 125I-pindolol binding, was increased two- to threefold by a 24-h incubation of the cells with IL-1 alpha, IL-1 beta, and TNF-alpha (EC50: 2.7, 8.2, and 24 pM, respectively), although a series of other cytokines and growth factors did not have this effect. Cortisol also increased beta AR density (EC50: 30 nM) and markedly potentiated the effects of IL-1 alpha, IL-1 beta, and TNF-alpha. Neither IL-1 nor cortisol influenced the proportion of cell surface vs internalized beta AR. The IL-1-induced increase in beta AR density was half-maximal after 6 h, was reversible at a similar rate, and was blocked by 1 microM of cycloheximide. The effect of IL-1 on beta AR was specific, as the density of glucocorticoid receptors, measured by 3H-dexamethasone binding, was reduced by IL-1. Both cortisol and IL-1 potentiated the isoproterenol-induced increase in cAMP accumulation. IL-1 inhibited cell proliferation and thymidine uptake, and increased the adherence of A549 cells to the plastic culture flask, as quantified by a cell detachment assay. The effect of IL-1 on cell adherence was not inhibited by cycloheximide. Cortisol decreased cell adherence and prevented the IL-1-induced increase in adherence. The results indicate that multiple effects of IL-1 in a cultured tumor cell line involve different mechanisms, suggesting heterogeneity of IL-1R and/or coupling of IL-1R to distinct, nuclear, and nonnuclear, effector pathways.     

Identification of a high-affinity receptor for interleukin-1 beta in rat brain

A single type of high-affinity binding sites for IL-1 beta was identified in the rat hypothalamus (Kd = 1.0 +/- 0.2 nM) and cerebral cortex (Kd = 1.3 +/- 0.2 nM), but not in the pituitary. The maximum binding capacity (Bmax) in the hypothalamus (Bmax = 75.4 +/- 10.8 fmol/mg protein) was 4 times greater than in the cerebral cortex (Bmax = 17.2 +/- 1.5 fmol/mg protein). Neither various neuropeptides nor IL-2 appeared to influence the binding of [125I]IL-1 beta to the hypothalamic membrane preparations. The potency of unlabeled IL-1 alpha to replace the binding of [125I]IL-1 beta to the hypothalamic membrane preparations was considerably less than that of unlabeled IL-1 beta. These findings indicate that IL-1 beta receptors are heterogeneously distributed in the central nervous system and that IL-1 alpha does not bind with IL-1 beta receptors in the brain.     

Alpha1 adrenoceptor subtypes in human urinary bladder: sex and regional comparison

A detailed study of the presence of alpha1 AR binding sites and alpha1 AR subtype mRNA expression in human urinary bladder areas involved in the micturition (i.e. detrusor, trigone and neck) is reported here, investigating whether or not there are differences between sexes. Results obtained indicated that alpha1 AR proteins were detectable in each bladder area. In both sexes, the detrusor and the neck expressed similar levels of alpha1 ARs: respectively, detrusor: 14.6 +/- 1.2 in men and 13.1 +/- 1.1 fmol/mg prot in women; neck: 16.9 +/- 3.2 in men and 17.5 +/- 4.1 fmol/mg prot in women. In the trigone, significantly higher alpha1ARs were found in women compared to men (20.6 +/- 1.1 vs 11.7 +/- 0.7 fmol/mg prot). Subtype analysis indicated that in women, each area was endowed with mRNA encoding for each alpha1 AR subtype. The men detrusor expressed alpha1a and alpha1d ARs, while in the trigone and the neck, each subtype was present. Since the detrusor muscle hypertrophy is a marker of bladder obstructive outlet, the selective alpha1 AR subtype targeting arouses much interest, as evidence indicates that there are differences in signalling pathways among the subtypes. Furthermore, the significance of the alpha1 ARs coexpression is still unknown; interestingly, recent papers demonstrate that alpha1 AR subtypes could dimerize. Thus, in the human urinary bladder it may be suggested a potential level of alpha1 AR complexity that could have an impact on drug development.     

应激大鼠肾胞液[^3H]醛固酮结合活性的变化及调节机制的初步研究

为探讨烫伤引起病理性应激时大鼠肾胞液醛固酮结合活性的变化及可能的调节机制,以[～3H]-醛固酮为配体,用放射性配基-受体结合分析法测定了正常对照组、轻度烫伤组和重度烫伤组大鼠肾胞液醛固酮结合活性的结合容量(Rt)和表观解离常数(Kd);采用体内注射TNF-α、IL-1β中和抗体和α-促黑色素细胞刺激激素(α-melanocyte-stimulatinghormone,α-MSH)和合成肽KPV(Ac-D-Lys-L-Pro-D-Val)等措施调节其改变.结果发现,肾胞液存在两种不同结合容量、不同亲和力的醛固酮结合活性受体.与正常对照组的Rt(Rt141.6±7.2fmol/mgpro;Rt2317.6±70.0fmol/mgpro)相比,轻烫组的Rt(Rt141.4±5.0fmol/mgpro;Rt2314.8±45.7fmol/mgpro)无显著差异(P>0.05;P>0.05);重烫组的Rt(Rt122.4±5.4fmol/mgpro;Rt2196.3±32.5fmol/mgpro)则显著下降(P<0.01;P<0.01).体内注射TNF-α与IL-1β中和抗体、α-MSH及KPV均能明显提高重烫组Rt值.结果提示,重度烫伤大鼠肾胞浆醛固酮结合活性降低,TNF-α、IL-1β中和抗体、α-MSH及KPV均可防止重度烫伤引起病理性应激时醛固酮结合活性降低.     

Synergistic regulation of pulmonary beta-adrenergic receptors by glucocorticoids and interleukin-1

Coculturing IM9 human lymphocytes and A549 human lung adenocarcinoma cells results in a 2-3-fold increase in the density of beta-adrenergic receptors in the latter, as quantified by 125I-cyanopindolol binding. Lymphocyte-conditioned medium (LCM) has the same effect, which is moderately sensitive to heat, is retained by ultrafiltration over a Mr 10,000 cut-off filter, and is reduced by trypsin treatment or by preincubation of lymphocytes with 0.3 micrograms/ml cycloheximide. Treatment of lung cells with cycloheximide also prevents the effect of LCM. Glucocorticoids, which also increase beta-receptor density in A549 cells, markedly potentiate the effect of LCM. Gel permeation high pressure liquid chromatography of LCM yields three peaks of biological activity with Mr 70,000, 35,000, and 15,000. Monocytic interleukin-1 (IL-1) mimics the effect of LCM in that it increases beta-receptor density in A549 cells (EC50 0.3 pM), and its effect is potentiated by cortisol. Recombinant IL-1 alpha is somewhat more potent than IL-1 beta, while interleukin-2 and interferon-alpha are ineffective. Tumor necrosis factor alpha causes a small increase in beta-receptors, which is not influenced by glucocorticoids. A polyclonal anti-IL-1 antibody inhibits the effect of IL-1 and the effect of the 15-kDa but not the 35- and 70-kDa fractions of LCM. The activity of the latter two fractions is also unaffected by anti-tumor necrosis factor alpha antibody. These results indicate that lymphocytes release protein factors including IL-1 that up-regulate pulmonary beta-adrenergic receptors by an action that involves protein synthesis. The possible relevance of this regulatory mechanism for the pathomechanism of certain respiratory diseases is discussed.     

Interleukin-1 inhibits PGE2 binding to macrophage-like P388D1 cells by a cyclic AMP-independent process.

The interaction between interleukin IL-1 alpha and PGE2 on P388D1 cells has been investigated. Preincubation of murine macrophage-like cells, P388D1, with IL-1 alpha (0-73 pM) reduced the binding of PGE2 to these cells in a concentration-dependent manner. Scatchard analysis showed that IL-1 alpha decreased the PGE2 binding by lowering both the high and low affinity receptor binding capacities (from 0.31 +/- 0.02 to 0.12 +/- 0.01 fmol/10(6) cells for the high affinity receptor binding sites and from 2.41 +/- 0.12 to 1.51 +/- 0.21 fmol/10(6) cells for the low affinity receptor binding sites). However, the dissociation constants of the receptors of the IL-1 alpha-treated cells remained unchanged. Inhibition of PGE2 binding by IL-1 alpha did not involve changes in either protein phosphorylation or intracellular cyclic AMP levels. Our data clearly show that IL-1 alpha inhibits the binding of PGE2 to monocytes/macrophages and may thereby counter the immunosuppressive actions of PGE2.     

Rapid inverse changes in alpha 1B- and beta 2-adrenergic receptors and gene transcripts in acutely isolated rat liver cells.

In vitro incubation of hepatocytes acutely isolated from adult male rats leads to a rapid conversion of the adrenergic activation of glycogenolysis from an alpha 1-receptor (alpha 1AR) to a beta 2-receptor (beta 2AR) mediated response within 4 h. In order to understand the underlying mechanism, we examined time-dependent changes in alpha 1- and beta 2-adrenergic activation of glycogenolysis and second messenger systems, the cellular density and affinity of alpha 1AR and beta 2AR, and the steady state levels of alpha 1BAR and beta 2AR mRNAs. Incubation of hepatocytes for 4 h resulted in a decrease in phosphorylase activation and inositol 1,4,5 trisphosphate accumulation in response to phenylephrine, a 40% decrease in alpha 1AR density, and a 70% decrease in alpha 1BAR mRNA levels. Incubation of hepatocytes for 4 h also resulted in the emergence of a phosphorylase response to isoproterenol, an increase in isoproterenol-induced but not in glucagon- or forskolin-induced cAMP accumulation, no significant change in beta 2AR density, and a twofold increase in beta 2AR mRNA levels. Exposure of cells to cycloheximide, 2 microM throughout the 4 h incubation, prevented the emergence of the phosphorylase response to isoproterenol and reduced beta 2AR densities, while the decrease in alpha 1AR density was not affected and the decrease in phosphorylase activation by phenylephrine was attenuated. The results indicate that dissociation of rat liver cells triggers a rapidly developing decrease in alpha 1BAR mRNA and increase in beta 2AR mRNA levels and corresponding inverse changes in the synthesis of alpha 1BAR and beta 2AR which account, at least in part, for the rapid conversion from alpha 1- to beta 2-adrenergic glycogenolysis.     

Receptors for prostaglandins F2 alpha and E2 in ovine corpora lutea during maternal recognition of pregnancy.

Plasma membrane receptors for prostaglandins (PG) F2 alpha and E2 were quantified in ovine corpora lutea obtained from nonpregnant and pregnant ewes on Days 10, 13, and 15 post-estrus, and from additional ewes on Days 25 and 40 of pregnancy. Regardless of reproductive status or day post-estrus, concentrations of luteal receptors for PGF2 alpha were 7- to 10-fold greater than those for PGE2. In pregnant ewes the concentration of receptors for PGF2 alpha was highest on Day 10 (35.4 +/- 2.8 fmol/mg) and lowest on Day 25 (22.3 +/- 2.5 fmol/mg). A difference in the concentration of luteal receptors for PGF2 alpha between pregnant and nonpregnant ewes was apparent only on Day 15 post-estrus, at which time the concentration of receptors for PGF2 alpha was higher in pregnant ewes than in nonpregnant ewes (27.1 +/- 2.7 vs. 17.7 +/- 2.7 fmol/mg). Concentrations of receptors for PGE2 in pregnant ewes were similar (p > 0.05; 2.8 +/- 0.3 to 3.7 +/- 0.2 fmol/mg) between Days 13 and 40 but were higher (p < 0.05) than in corpora lutea obtained from nonpregnant ewes on Days 10 (5.0 +/- 0.4 vs. 4.1 +/- 0.2 fmol/mg) and 15 (3.7 +/- 0.2 vs. 2.0 +/- 0.4 fmol/mg) post-estrus. Although concentrations of receptors for both PGF2 alpha and PGE2 were lowest in corpora lutea obtained from nonpregnant ewes on Day 15, this was not due to luteal regression since the weights and concentrations of progesterone in corpora lutea on Day 15 were not lower than those for corpora lutea obtained on Days 10 and 13.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of interleukin 8 (IL-8) production by Pseudomonas nitrite reductase in human alveolar macrophages and epithelial cells.

Interleukin-8 (IL-8) participates in the generation of dense neutrophil accumulations in bronchopulmonary infections caused by Pseudomonas aeruginosa (P. aeruginosa). We have recently reported that nitrite reductase, a bifunctional enzyme located in the periplasmic space of P. aeruginosa, induces IL-8 generation in bronchial epithelial cells (K. Oishi et al. Infect. Immun. 65: 2648-2655, 1997). We examined whether or not Pseudomonas nitrite reductase (PNR) could also stimulate human alveolar macrophages (AM) and pulmonary type II epithelial-like cells (A549) to induce IL-8 production and mRNA expression as well as the production of TNF alpha and IL-1beta. We demonstrated a time- and dose-dependent IL-8 protein synthesis and IL-8 mRNA expression, but no TNF alpha or IL-1beta production, by A549 cells in response to PNR. New protein translation was not required for PNR-mediated IL-8 mRNA expression in the same cells. Furthermore, simultaneous stimulation of PNR with serial doses of TNF alpha or IL-1beta resulted in additive IL-8 production in A549 cells. In adherent AM, PNR enhanced IL-8 protein synthesis and IL-8 mRNA expression in a time-dependent fashion. PNR similarly induced a time-dependent production of TNF alpha and IL-1beta by human adherent AM. Neutralization of TNF alpha or IL-1beta did not influence the levels of IL-8 production in adherent AM culture. We also evaluated whether the culture supernatants of the A549 cells or AM stimulated with PNR could similarly mediate neutrophil migration in vitro. When anti-human IL-8 immunoglobulin G was used for neutralizing neutrophil chemotactic factor (NCF) activities in the culture supernatants of these cells stimulated with 5 microg/ml of PNR, the mean percent reduction of NCF activities were 49-59% in A549 cells and 24-34% in AM. Our present data support that PNR directly stimulates AM and pulmonary epithelial cells to produce IL-8. PNR also mediates neutrophil migration, in part, through IL-8 production from AM and pulmonary epithelial cells. These data suggest the contribution of PNR to the pathogenesis of bronchopulmonary infections due to P. aeruginosa.     

Preponderance of alpha 2- over beta 1-adrenergic receptor sites in human fat cells is not predictive of the lipolytic effect of physiological catecholamines

Adrenergic control of human fat cell lipolysis is mediated by two kinds of receptor sites that are simultaneously stimulated by physiological amines. To establish a correlation between the binding characteristics of the receptor and biological functions, the ability of physiological amines to stimulate or inhibit isolated fat cell lipolysis in vitro was compared to the beta- and alpha 2-adrenoceptor properties of the same fat cell batch. The beta-selective antagonist (-)[3H]dihydroalprenolol ([3H]DHA) and the alpha 2-selective antagonists [3H]yohimbine ([3H]YOH) and [3H]rauwolscine ([3H]RAU) were used to identify and characterize the two receptor sites. Binding of each ligand was rapid, saturable, and specific. The results demonstrate 1) the weaker lipolytic effect of epinephrine compared with norepinephrine. This can be explained by the equipotency of the amines at the beta 1-sites and the higher affinity of epinephrine for alpha 2-adrenergic receptors. 2) The preponderance of alpha 2-adrenergic receptor sites labeled by [3H]YOH (Bmax, 586 +/- 95 fmol/mg protein; KD, 2.7 +/- 0.2 nM) or [3H]RAU (Bmax, 580 +/- 100 fmol/mg protein; KD, 3.7 +/- 0.1 nM). These two ligands can be successfully used to label alpha 2-adrenergic receptor sites. 3) The beta 1-adrenergic receptor population labeled by [3H]DHA(Bmax, 234 +/- 37 fmol/mg protein; KD, 1.8 +/- 0.4 nM), although a third as numerous as the alpha 2-adrenergic population, is responsible for the lipolytic effect of physiological amines and is weakly counteracted by simultaneous alpha 2-adrenergic receptor stimulation under our experimental conditions. It is concluded that, in human fat cells, the characterization of beta 1- and alpha 2-adrenergic receptors by saturation studies or kinetic analysis to determine affinity (KD) and maximal number of binding sites (Bmax) is not sufficient for an accurate characterization of the functional adrenergic receptors involved in the observed biological effect.     

α<Superscript>5</Superscript>β<Subscript>1</Subscript> integrins and fibronectin are involved in adherence of the<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ER97314 clinical strain to A549 cells

The role of fibronectin (Fn) and its natural receptors alpha5beta1 integrins in the interaction of P. aeruginosa with A549 epithelial cells was compared in the clinical isolate ER97314 and the reference PAK strain. Both strains expressed functional type IV pili, as shown by the results of the twitching motility assay. The ER97314 strain was highly adherent to immobilized Fn (640 000+/-20 000 CFU per well) while the PAK strain adhered less efficiently (70 000+/-10 000 CFU per well). Both strains adhered to A549 cells (33 400+/-1200 and 1200+/-100 CFU per well, for PAK and ER97314, respectively), only the PAK strain being significantly internalized (9430+/-2020 CFU per well). Cytochalasin D and genistein significantly decreased bacterial adherence of the 2 strains and caused also a significant decrease in PAK internalization. This inhibitory activity was not related to changes in the expression of alpha5beta1 integrins. Antibodies to Fn and alpha5beta1 integrins inhibited the adherence of the ER97314 strain but had no significant effect on PAK interaction with human cells. These findings suggest that only some P. aeruginosa strains can target Fn and their natural receptors alpha5beta1 integrins for adherence to A549 cells.     

Presence of high affinity receptor for interleukin-1 (IL-1) on cultured porcine thyroid cells

Using 125I-interleukin-1 beta (125I-IL-1 beta) as a ligand, a specific receptor of high affinity dissociation constant (1.1 +/- 0.2 x 10(-10) M) with binding sites (350 +/- 40/cell) for interleukin-1 beta (IL-1 beta) has been demonstrated on cultured porcine thyroid cells. IL-1 alpha almost equally cross-reacted with the receptor (Kd = 1.2 +/- 0.3 x 10(-10) M and 350 +/- 50 binding sites/cell). TSH, IL-2 and other peptide hormones did not inhibit the binding of 125I-IL-1 beta to thyroid cells. Crosslinking study revealed a major band (approximately 95 kD) with a corrected molecular mass of approximately 78 kD. Moreover, both IL-1 beta and IL-1 alpha stimulated prostaglandin E2 production of cultured porcine thyroid cells, although the potency of IL-1 alpha was slightly greater than that of IL-1 beta. These results suggest that IL-1 may be involved in the regulation of thyroid cell function.     

Estrogen and androgen receptors in the liver of cynomolgus monkeys (Macaca fascicularis)

Estrogen receptors (ER) and androgen receptors (AR) were evaluated in the hepatic cytosol from cynomolgus macaques to determine if there were differences associated with gender and endogenous hormone secretion. Saturable, high affinity binding (Kd = 0.2-0.8 nM) was demonstrated for both ER and AR from either male or female monkeys. Displacement of tritiated estradiol from the ER was estrogen specific (including ethinyl estradiol). Both androgens and the synthetic progestins (levonorgestrel and norethindrone) displaced tritiated mibolerone from the AR. Both 8S and 4S molecular forms of ER and AR were demonstrated on 5-20% sucrose density gradients. The ER levels were higher in females in the follicular phase of the menstrual cycle (40.5 +/- 1.9 fmol/mg protein) than levels in males (26.4 +/- 4.8 fmol/mg protein; P less than 0.01) or levels in luteal phase females (31.8 +/- 2.4 fmol/mg protein; P less than 0.05). AR levels were not different between females during different phases of the menstrual cycle (65.8 +/- 4.6 and 69.5 +/- 4.3 fmol/mg protein, follicular and luteal, respectively), but there was a tendency (P less than 0.10) for the levels in males (54.4 +/- 6.6 fmol/mg protein) to be lower than female levels. The demonstration of saturable, high affinity binding of androgens and estrogens in liver tissue of these primates, along with differences associated with gender and the stage of the menstrual cycle, suggests that hepatic receptors are functional and may play an important role in hepatic protein secretion.     

Fusion of beta 2-adrenergic receptor to G alpha s in mammalian cells: identification of a specific signal transduction species not characteristic of constitutive activation or precoupling

The forward and antegrade interactions that comprise the agonist receptor-G protein complex were studied in Chinese hamster fibroblasts transfected to express the beta(2)-adrenergic receptor (beta(2)AR), the beta(2)AR and the alpha-subunit of its cognate G protein (G(s)), and a protein consisting of the beta(2)AR fused at its carboxy terminus with G(alpha)(s) (beta(2)AR-G(s)). Expression levels were matched at approximately 600 fmol/mg. Basal adenylyl cyclase activities were increased with the fusion receptor membranes compared to coexpressed receptor plus G(alpha)(s), and to wild-type beta(2)AR (20.5 +/- 1.8 vs 9.0 +/- 0.88 vs 8.7 +/- 0.93 pmol min(-)(1) mg(-)(1)), confirming in mammalian cells that the fusion of beta(2)AR and G(alpha)(s) results in a state not attained by expression of unfused components. However, agonist-stimulated activities were not increased proportionally, such that the stimulation over basal of the beta(2)AR-G(s) fusion protein (1. 5-fold) was less than wild-type beta(2)AR (2.1-fold). Agonist competition studies performed in the absence of guanine nucleotide exhibited high-affinity binding sites with a lower K(H) (1.75 vs 8. 47 nM) and greater %R(H) (51% vs 44%) for beta(2)AR-G(s), but GppNHp failed to convert most of these to the low-affinity state. Functional studies with the inverse agonist ICI 118551 did not show enhanced efficacy or potency with the fusion protein. Adenylyl cyclase studies with three partial agonists with diverse structures (dobutamine, ritodrine, and phenylephrine) showed no enhancement of efficacy with beta(2)AR-G(s) and a minor trend toward enhanced potency. Taken together, these results indicate that the tethering of G(alpha)(s) to the beta(2)AR causes a conformational change in the receptor that stabilizes a species "trapped" between the non-guanine nucleotide-bound state and the GTP-bound form. Functionally the receptor is not characterized by a consistent pattern of properties ascribed to other states such as constitutive activation or precoupling, but rather represents a unique state in the transition from high- to low-affinity forms.     

Cross-regulation between G-protein-coupled receptors. Activation of beta 2-adrenergic receptors increases alpha 1-adrenergic receptor mRNA levels.

Stimulation of DDT1 MF-2 vas deferens cells with epinephrine resulted in a time- and dose-dependent loss of alpha 1-adrenergic receptor-specific ligand binding. Regulation of alpha 1-adrenergic receptor mRNA was characterized. In monolayer culture, cells displayed 0.7 +/- 0.05 amol of alpha 1-adrenergic receptor mRNA/microgram of total cellular RNA. Epinephrine, which acts at both alpha 1- and beta 2-adrenergic receptors of DDT1 MF-2 cells, induced a short term (2-8 h) increase (50-70%) in the abundance of alpha 1-adrenergic receptor mRNA. Propranolol, a beta 2-adrenergic receptor antagonist, attenuated the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA but did not affect the decrease in alpha 1-adrenergic receptor-specific ligand binding. Phentolamine, an alpha 1-adrenergic receptor antagonist, did not attenuate the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA at 4 h but did block the decrease in alpha 1-adrenergic receptor-specific ligand binding. The half-life of the alpha 1-adrenergic receptor mRNA was approximately 7 h in untreated cells as well as in cells challenged with epinephrine. The epinephrine-promoted increase in alpha 1-adrenergic receptor mRNA was found to result from cross-regulation via beta 2-adrenergic receptors. Cholera toxin, forskolin, as well as the cyclic AMP analog CPT cAMP (8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate) increased the alpha 1-adrenergic receptor mRNA at 4 h, as did epinephrine in the presence of alpha 1-antagonists but not in the presence of a beta-adrenergic antagonist. This is the first report of heterologous up-regulation of mRNA levels of adrenergic receptors. Cross-regulation between alpha 1- and beta 2-adrenergic receptor-mediated pathways at 4 h occurs at the level of mRNA whereas later down-regulation of alpha 1-receptor mRNA and binding proceed via agonist activation of alpha 1-adrenergic receptors.     

Binding of the hydrophilic beta-adrenergic antagonist [3H]CGP-12177 to cardiac tissue slices: characterization and ontogenetic studies in dogs

A new technique was developed to characterize the binding of a hydrophilic beta-adrenergic antagonist, [3H]CGP-12177, to 1-mm thick slices of canine cardiac tissue. This technique was used to quantify the density (Bmax) and the affinity (Kd) of these receptors in the right ventricular conus (RVC) and the left ventricle (LV) at day 1 to 6 weeks of age, and in the adult. Binding was found to be reversible, saturable, stereospecific, of high affinity, and thermolabile. There was an increase in the density of beta-adrenergic receptors between day 1 (Bmax = 2.2 +/- 0.3 fmol/mg tissue in RVC and 2.9 +/- 0.8 fmol/mg tissue in the LV) and 2 weeks of age postnatally, after which it remained constant until 6 weeks of age (Bmax = 7.5 +/- 0.4 and 6.8 +/- 0.9 fmol/mg tissue in RVC and LV, respectively); however, by 6 weeks of age it had not reached adult levels (10.3 +/- 1.0 fmol/mg tissue). The affinity of these receptors did not change between early neonatal life (Kd = 1.3 +/- 0.4 nM) and adulthood (Kd = 1.4 +/- 0.2 nM). The density of beta-adrenergic receptors in the RVC was similar to that in the LV. This new method of quantifying beta-adrenergic receptors in cardiac tissue is simple and fast, and requires minimal tissue handling. It proved to be useful in studying the development of cardiac beta-adrenergic receptors with age.     

Interleukin-1beta stimulates macrophage inflammatory protein-1alpha and -1beta expression in human neuronal cells (NT2-N)

Chemokines are important mediators in immune responses and inflammatory processes of neuroimmunologic and infectious diseases. Although chemokines are expressed predominantly by cells of the immune system, neurons also express chemokines and chemokine receptors. We report herein that human neuronal cells (NT2-N) produce macrophage inflammatory protein-1alpha and -1beta (MIP-1alpha and MIP-1beta), which could be enhanced by interleukin (IL)-1beta at both mRNA and protein levels. The addition of supernatants from human peripheral blood monocyte-derived macrophage (MDM) cultures induced MIP-1beta mRNA expression in NT2-N cells. Anti-IL-1beta antibody removed most, but not all, of the MDM culture supernatant-induced MIP-1beta mRNA expression in NT2-N cells, suggesting that IL-1beta in the MDM culture supernatants is a major factor in the induction of MIP-1beta expression. Investigation of the mechanism(s) responsible for IL-1beta-induced MIP-1alpha and -1beta expression demonstrated that IL-1beta activated nuclear factor kappa B (NF-kappaB) promoter-directed luciferase activity in NT2-N cells. Caffeic acid phenethyl ester, a potent and specific inhibitor of activation of NF-kappaB, not only blocked IL-1beta-induced activation of the NF-kappaB promoter but also decreased IL-1beta-induced MIP-1alpha and -1beta expression in NT2-N cells. These data suggest that NF-kappaB is at least partially involved in the IL-1beta-mediated action on MIP-1alpha and -1beta in NT2-N cells. IL-1beta-mediated up-regulation of beta-chemokine expression may have important implications in the immunopathogenesis of inflammatory diseases in the CNS.