Effect of siderophores, catecholamines, and catechol compounds on Listeria spp. Growth in iron-complexed medium

The objective of our study was to elucidate the involvement of interleukin (IL)-8 in the hCG-induced rabbit ovulatory process. After administering hCG (100 IU i.v.), we examined myeloperoxidase (MPO) activity, which represents neutrophil accumulation; neutrophil elastase (NE) activity, which is an indicator of neutrophil activity; and levels of IL-8 in the ovaries. The maximal level of IL-8 was observed before MPO and NE activities reached a peak: production of IL-8, MPO, and NE activities peaked, respectively, at 4 h (5.58 +/- 0.88 pg/mg ovary, n = 13), 6 h (1.07 +/- 0.13 deltaA/min per gram ovary, n = 8), and 9 h (18.89 +/- 1.05 U/g ovary, n = 8). Anti-rabbit IL-8 antiserum given i.v. significantly reduced the maximal levels of hCG-induced MPO activity (antiserum vs. control; 0.34 +/- 0.04 vs. 1.12 +/- 0.11 deltaA/min per gram ovary, n = 14, p < 0.001) and NE activity (8.14 +/- 0.85 vs. 18.30 +/- 0.79 U/g ovary, n = 14, p < 0.001). The hCG-induced ovulation rate was significantly inhibited by the antiserum (50.5% vs. 83.9%, n = 14, p < 0.001). Intraperitoneal injection of 100 mg/kg of ONO-5046, a specific NE inhibitor, also attenuated the ovulation rate (ONO-5046 vs. vehicle; 56.0% vs. 74.0%, n = 14, p < 0.05). These findings clearly indicate that IL-8 has an important role in the hCG-induced ovulatory process through the accumulation and activation of neutrophils.     

Increased interleukin-1 and interleukin-1 receptor antagonist levels in cervical mucus in the ovulatory phase in comparison with the follicular phase

Interleukin-1 (IL-1) receptor antagonist (IL-1ra) levels in the cervical mucus of women in the ovulatory phase are significantly higher than those in the follicular phase. IL-1 titers of women in the ovulatory phase are also significantly higher than those in the follicular phase. A positive correlation between IL-1ra and IL-1 levels in the cervical mucus was observed. Immunohistochemistry using an anti-IL-1ra monoclonal antibody revealed positive staining in the epithelial cells of the endocervix. These results suggest that IL-1ra from cervical epithelial cells protects the reproductive system from the toxicity of IL-1 produced in the endocervix.     

A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance

In the present study, we analyzed the cytokine network among TNFalpha, IL-1beta, IL-8, and IL-1 receptor antagonist (IL-1Ra) in a rabbit experimental model of acute gout. The production of TNFalpha in synovial fluids reached the peak at 2 hours after the intra-articular injection of monosodium urate (MSU) crystals. The production of IL-1beta and IL-8 reached the first peak at 2 hours and the second peak at 9 and 12 hours, respectively. The production of endogenous IL-1Ra reached the peak at 9 hours. The source of TNFalpha and the first phase of IL-8 was synovial cells, whereas infiltrating leukocytes were the source of the second phase of IL-8 and also of IL-1beta and IL-1Ra. The production of TNFalpha was not altered by either anti-lL-8 IgG or IL-1Ra. The first IL-1beta peak was reduced only with a combination of anti-TNFalpha mAb and anti-lL-8 IgG, whereas the second peak was significantly reduced by either inhibitor. The first IL-8 peak was not altered with anti-TNFalpha mAb or IL-1 Ra, whereas the second IL-8 peak was reduced with IL-1Ra. Anti-TNFalpha mAb or anti-lL-8 IgG significantly reduced the peak level of endogenous IL-1Ra. These cytokine inhibitors also attenuated the maximal leukocyte accumulation at 9 hours, but not the initial phase, which occurred within 2 hours. These results provide evidence that IL-8 and TNFalpha were responsible for the production of IL-1beta and IL-1Ra, and that IL-1beta was responsible for the second phase of IL-1beta and IL-8 production. Our data also suggest that the initial and the maximal phases of leukocyte influx are differently regulated. Finally, the intravenous injection of colchicine inhibited neutrophil infiltration without affecting the production of TNFalpha or the first peak of IL-8, suggesting that colchicine inhibits MSU crystal-induced arthritis by directly inhibiting the migration of neutrophils.     

Generation and characterization of a human monoclonal autoantibody that acts as a high affinity interleukin-1 alpha specific inhibitor

Interleukin-1 (IL-1) defines two polypeptides, IL-1 alpha and IL-1 beta, that possess a wide spectrum of biological effects. Two natural antagonists of IL-1 action have been characterized: the IL-1 receptor antagonist (IL-1Ra) and a soluble form of the type II IL-1 receptor. Neutralizing autoantibodies to IL-1 alpha have also been detected in sera of healthy individuals and patients with autoimmune or inflammatory diseases. To characterize such antibodies molecularly, we attempted to generate B cell clones producing anti-IL-1 alpha human monoclonal antibody (HuMAb) by combining Epstein-Barr virus-immortalization and CD40-activation of B lymphocytes from individuals with circulating anti-IL-1 alpha. We describe herein the generation and properties of a natural IgG4/kappa anti-IL-1 alpha monoclonal autoantibody, HuMAb X3, that bound specifically to human IL-1 alpha, but not to IL-1 beta and IL-1Ra, with a high affinity (Kd = 1.2 x 10(-10)M). HuMAb X3 inhibited IL-1 alpha binding to IL-1 receptors and neutralized biological activities of both recombinant and natural forms of IL-1 alpha. A recombinant form of HuMAb X3 was found to display identical specific IL-1 alpha antagonism. The presence of somatic mutations within X3 variable regions suggests an antigen-driven affinity maturation. This study extends the demonstration of the presence of high affinity neutralizing anti-IL-1 alpha autoantibodies that can function as a third type of IL-1 antagonist.     

Differential expression of two major cytokines produced by neutrophils, interleukin-8 and the interleukin-1 receptor antagonist, in neutrophils isolated from the synovial fluid and peripheral blood of patients with rheumatoid arthritis

OBJECTIVE: Neutrophils have been shown to produce interleukin-8 (IL-8) and interleukin-1 receptor antagonist (IL-1ra) in large amounts compared with other cytokines. Since IL-8 has a proinflammatory action whereas IL-1ra is antiinflammatory, our objective was to examine the relative levels of production of these cytokines by synovial fluid (SF) neutrophils isolated from patients with rheumatoid arthritis (RA). METHODS: We measured cytokine production using an enzyme-linked immunosorbent assay and analyzed messenger RNA accumulation in cells by Northern blot. RESULTS: SF neutrophils produced significantly more IL-8 and IL-1 beta, but significantly less IL-1ra, than peripheral blood neutrophils. CONCLUSION: These observations provide new information on the production of pro- and antiinflammatory molecules by neutrophils in the SF environment, and their possible role in RA.     

Production and regulation of monocyte chemoattractant protein-1 in lipopolysaccharide- or monosodium urate crystal-induced arthritis in rabbits: roles of tumor necrosis factor alpha, interleukin-1, and interleukin-8

The production of monocyte chemoattractant protein-1 (MCP-1) and its regulation by TNFalpha, IL-1, and IL-8 were investigated in two rabbit models of arthritis induced by intra-articular injection of lipopolysaccharide (LPS) or monosodium urate (MSU) crystals. We first prepared recombinant rabbit MCP-1 and antibodies and then developed an immunoassay. The immunoassay detected 3 pg/ml rabbit MCP-1 and did not cross-react with other rabbit chemokines such as IL-8 or GRO. MCP-1 was first detected in synovial fluid (SF) at 1 hour, and peaked at 4 or 2 hours after the injection of LPS or MSU crystals, respectively. Immunohistochemically, MCP-1 was detected in synovial lining cells and infiltrating neutrophils. The amounts of MCP-1 detected in SF from neutrophil-depleted rabbits were similar to those in normal rabbits, suggesting that synovial lining cells were the main source of MCP-1 detected in SF. The peak level of MCP-1 in SF after LPS-injection was inhibited by 57% with anti-TNFalpha mAb and by 41% with IL-1 receptor antagonist (IL-1Ra). Coadministration of anti-TNFalpha mAb and IL-1Ra inhibited 90% of MCP-1 production. In contrast, the peak level of MCP-1 in SF after MSU crystal-injection was not affected by any cytokine inhibitor, but was reduced by 52% with coadministration of anti-TNFalpha mAb and IL-1Ra. Anti-IL-8 IgG had no effect on the production of MCP-1 in either model. Thus, the production of MCP-1 in LPS-induced arthritis was mostly regulated by TNFalpha and IL-1, whereas half the extent of MCP-1 production in MSU crystal-induced arthritis was independent of TNFalpha or IL-1. IL-8 does not seem to regulate the production of MCP-1 in SF either directly or indirectly. Finally, administration of neutralizing anti-MCP-1 antibody inhibited LPS- and MSU crystal-induced monocyte infiltration by 58.4% and 44.9%, respectively, suggesting that synovial production of MCP-1 plays an important role in the recruitment of monocytes in these arthritis models.     

A decomposition product of a contaminant implicated in L-tryptophan eosinophilia myalgia syndrome affects spinal cord neuronal cell death and survival through stereospecific, maturation and partly interleukin-1-dependent mechanisms

The L-tryptophan eosinophilia myalgia syndrome (L-TRP-EMS), an inflammatory syndrome characterized by eosinophilia, myalgias, perimyositis, fasciitis and neuropathies, occurred in epidemic proportions in the United States in the summer and fall of 1989. The neuropathic clinical features in L-TRP EMS are complex and mixed. In the present study, one of the impurities most highly associated with development of L-TRP EMS, 1,1'-ethylidenebis[L-tryptophan] (EBT), and two of its diastereoisomeric breakdown products, were compared for evidence of neurotoxicity in vitro. In 1-month-old spinal cord cultures derived from fetal mice, synthetic (-)-(1S,3S)-1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (1S-beta-C) produced a 30 to 35% loss in numbers of neurons. Toxicity was not apparent after treatment with the R-isomer of the same compound or with the parent compound, EBT. Cotreatment of cultures with 1S-beta-C and neutralizing antiserum to interleukin-1 alpha (IL-1 alpha), or with 1S-beta-C and neutralizing antiserum against the murine IL-1 receptor, prevented neuronal cell death associated with 1S-beta-C. Recombinant IL-1 alpha also produced neuronal killing that was not additive to that observed with the 1S-beta-C treatment. In contrast, in immature spinal cord neuronal cultures, the 1S-beta-C, but not the 1R-beta-C or EBT, prevented the 30% cell death which normally occurs in these cultures. Neither neutralizing anti-IL-1 antibody, nor anti-IL-1 receptor antibody blocked the neuronal survival effect, suggesting that 1S-beta-C induces neuronal survival through a receptor-mediated mechanism independent of IL-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studying the mental health of a nation: a preliminary report on a population survey in Singapore

OBJECTIVE: Requirements for cytokines and adhesion molecules for peritoneal neutrophil recruitment during glycogen-induced peritonitis in rats were systematically defined. SUBJECTS: Male Long Evans rats (275-300 g). METHODS: Four hours after intraperitoneal injection of 25 mg oyster glycogen, neutrophilic exudates were harvested. Effects of blocking reagents (injected intravenously) to rat E-, L- and P-selectins, beta1 (VLA-4) and beta2 integrins (LFA-1 and Mac-1), ICAM-1, and the cytokines TNFalpha, IL- and IL-8 were assessed. RESULTS: Administration of synthetic sialyl Lewis(x) oligosaccharide reduced neutrophil recruitment to the peritoneum by 26%. Antibody to E-selectin reduced neutrophil accumulation by 71%, while anti-L-selectin reduced neutrophil accumulation by 59%, and anti-P-selectin was without an effect. Similar patterns of inhibition were found when selectin-Ig chimeras were employed. Antibodies to LFA-1 (CD11a), Mac-1 (CD11b) or CD18 reduced neutrophil accumulation by 62 percent, 59 percent and 86%, respectively, while anti-VLA-4 was without effect. Anti-ICAM-1 reduced cell influx by 65%. IL-1 receptor antagonist and antibodies to IL-1 and human IL-8 reduced neutrophil accumulation by 43alpha, 40% and 62 percent, respectively. Unexpectedly, blockade of TNFalpha had no effect. CONCLUSIONS: These studies identify requirements for selectins, beta2 integrins, IL-1 and a rat chemokine(s) similar to human IL-8 for neutrophil recruitment during glycogen-induced peritonitis. The lack of participation of VLA-4, P-selectin and TNFalpha suggests organ-specific cytokine and adhesion molecule requirements for neutrophil recruitment.     

The role of interleukin-6 in the activation of the hypothalamo-pituitary-adrenocortical axis and brain indoleamines by endotoxin and interleukin-1 beta

Interleukin-6 (IL-6) is one of several cytokines that can stimulate the hypothalamo-pituitary-adrenocortical (HPA) axis. Because IL-6 is produced in response to the administration of endotoxin (LPS) and interleukin-1 (IL-1), it is possible that IL-6 contributes to the neuroendocrine and neurochemical changes induced by them. In this study, intraperitoneal (i.p.) injection of LPS elevated plasma concentrations of IL-6 while activating the HPA axis in a dose-dependent manner. Both responses reached a peak at around 2-3 h. Mouse IL-1beta administration (100 ng, i.p.) induced large increases in plasma corticosterone and a substantial, but short-lived increase in plasma IL-6 with a peak at 2 h. Pretreatment of mice intraperitoneally with a monoclonal antibody to mouse IL-6 significantly attenuated the plasma ACTH and corticosterone responses to LPS at 3 h, but not at 1 h. Anti-IL-6 treatment also attenuated the LPS-induced increases of tryptophan and the serotonin catabolite, 5-hydroxyindoleacetic acid (5-HIAA), but not that of the norepinephrine catabolite, 3-methoxy,4-hydroxyphenylethyleneglycol (MHPG). Pretreatment of mice with anti-IL-6 significantly attenuated the IL-1-induced increases of plasma ACTH and corticosterone at 2 h, but not at 4 h. The IL-1-induced increases of MHPG, tryptophan and 5-HIAA in hypothalamus and brain stem were not significantly altered. These results suggest that IL-6 contributes to the later phases of the LPS- and IL-1-induced stimulations of the HPA axis and to the indoleaminergic responses to LPS, but not to IL-1.     

Systemic liberation of interleukin-1 beta and interleukin-1 receptor antagonist in the perioperative phase of liver transplantation

We measured systemic serum levels of interleukin-1 receptor antagonist (IL-1ra), interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), and interleukin-6 (IL-6) during the preoperative, anhepatic, and postreperfusional phases up to the 7th postoperative day in 60 patients undergoing orthotopic liver transplantation (LTx). In contrast to IL-1 beta, IL-1ra, TNF-alpha, and IL-6 showed a significant elevation in relation to the early phase after reperfusion, while TNF-alpha displayed a high grade of scatter. In addition, IL-1ra levels were significantly elevated during the anhepatic phase. Maximum serum levels were found at 15 min after reperfusion, 120 min after reperfusion, and on the 1st postoperative day, respectively. Serum levels decreased considerably at 24 h and 7 days after reperfusion. The comparative monitoring of systemic cytokine and cytokine antagonist levels, in particular the liberation of IL-1ra and IL-6 may provide useful parameters for the development of new liver preservation theories for LTx.     

A neuromodulatory role of interleukin-1beta in the hippocampus

It is widely accepted that interleukin-1beta (IL-1beta), a cytokine produced not only by immune cells but also by glial cells and certain neurons influences brain functions during infectious and inflammatory processes. It is still unclear, however, whether IL-1 production is triggered under nonpathological conditions during activation of a discrete neuronal population and whether this production has functional implications. Here, we show in vivo and in vitro that IL-1beta gene expression is substantially increased during long-term potentiation of synaptic transmission, a process considered to underlie certain forms of learning and memory. The increase in gene expression was long lasting, specific to potentiation, and could be prevented by blockade of potentiation with the N-methyl-D-aspartate (NMDA) receptor antagonist, (+/-)-2-amino-5-phosphonopentanoic acid (AP-5). Furthermore, blockade of IL-1 receptors by the specific interleukin-1 receptor antagonist (IL-1ra) resulted in a reversible impairment of long-term potentiation maintenance without affecting its induction. These results show for the first time that the production of biologically significant amounts of IL-1beta in the brain can be induced by a sustained increase in the activity of a discrete population of neurons and suggest a physiological involvement of this cytokine in synaptic plasticity.     

The morphogenic/cytotoxic and prostaglandin-stimulating activities of interleukin-1 beta in the rat ovary are nitric oxide independent

Nitric oxide (NO) has been implicated as a mediator of physiologic and pathologic cellular injury. Since the cytokine interleukin-1 beta (IL-1 beta) induces nitric oxide synthase (NOS) activity as well as effects morphogenic/cytotoxic changes and increased prostaglandin (PGE2) levels in cultured whole ovarian dispersates, we set out to determine whether these actions are interrelated. Treatment with IL-1 beta resulted in a marked increase in media nitrite and nitrate accumulation, morphological alterations, and increased release of lactate dehydrogenase (LDH) into media. Addition of IL-1 receptor antagonist (RA) eliminated these IL-1 beta effects. In contrast, specific inhibitors of NOS failed to reverse IL-1 beta-induced morphogenic changes or LDH release in spite of complete reduction of media nitrite to control levels. Similarly, treatment with transforming growth factor beta 1, inhibited IL-1 beta-induced nitrite accumulation, but had no effect on the morphologic or cytotoxic endpoints. Moreover, the addition of sodium nitroprusside, an NO generator, resulted in progressive increments in media nitrite content without a corresponding increase in the IL-1 beta-associated morphogenic changes or media LDH content. Furthermore, IL-1-induced PGE2 accumulation remained unaffected by specific NOS inhibition. These observations support the view that NO does not mediate the morphogenic/cytotoxic or inflammatory-like (e.g., PGE2 inducing) properties of IL-1 beta in cultured whole ovarian dispersates. Although the precise role of NO in ovarian physiology remains unknown, it is possible that NO participates in the periovulatory modulation of ovarian blood flow by virtue of its potent vasodilatory activity.     

Role of central interleukin-1 beta in gastrointestinal motor disturbances induced by lipopolysaccharide in sheep

Cytokines are involved in the symptoms of the acute phase response induced by infectious diseases in humans as well as in animals, and interleukin-1 beta (IL-1 beta) has a pivotal role in these changes. The role of central IL-1 beta in the gastrointestinal hypomotility and fever evoked by intravenous administration of lipopolysaccharide (LPS) and the mechanisms involved, were investigated in sheep as an experimental model. LPS (0.1 microgram/kg, intravenously) induced gastrointestinal hypomotility and fever that were significantly reduced by prior intracerebroventricular administration of IL-1 receptor antagonist protein (IL-1ra, 2 micrograms/kg). The effects of LPS were mimicked by intracerebroventricular IL-1 beta (50 ng/kg), whereas IL-1 beta injected intravenously at the same dose only caused a slight and transient fever without modifying the gastrointestinal motility. Prior intracerebroventricular administration of the cyclooxygenase inhibitor indomethacin (100 micrograms/kg) but not the corticotropin-releasing factor (CRF) receptor antagonist alpha-helical CRF9-41 (5 micrograms/kg) blocked all effects by both LPS and IL-1 beta. These results suggest that in sheep, LPS induces digestive motor disturbances through a central release of IL-1 beta and prostaglandins.     

Mode of action of interleukin-1 in suppression of pituitary LH release in castrated male rats

These studies were undertaken to elucidate the mechanisms whereby the cytokine, Interleukin (IL-1) suppresses pituitary LH release in orchidectomized rats. Since LH secretion is pulsatile in castrated rats, the effects of IL-1 on the components of the LH pulsatility were assessed. Intracerebroventricular (i.c.v.) administration of IL-1 alpha or IL-1 beta suppressed LH release, but IL-1 beta was relatively more effective than IL-1 alpha in terms of the onset (IL-1 beta = 30 min; IL-1 alpha = 105 min) as well as the magnitude and duration of LH suppression. Further, the marked suppression of LH secretion in IL-1 beta-treated rats was found to be due to significant reductions both in the frequency and amplitude of LH episodes. We next evaluated whether the IL-1 beta-induced suppression of LH release was mediated by either of the two inhibitory hypothalamic peptidergic systems, corticotrophin releasing hormone (CRH) and endogenous opioid peptides (EOP). Passive immunoneutralization of CRH by i.c.v. administration of a specific CRH-antibody, either once at 15 min or twice at 75 and 15 min before IL-1 beta injection, failed to block the suppressive effects of IL-1 beta on LH release. Similarly, pharmacological blockade of CRH by i.c.v. injection of the CRH receptor antagonist, alpha-helical CRH9-41 15 min before IL-1 beta was ineffective. However, i.v. infusion of the opiate receptor antagonist, naloxone, which on its own had no effect on LH secretion, counteracted the inhibitory effects of IL-1 beta. To further identify the opiate receptor subtype involved, we utilized specific opiate receptor subtype antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Does increased urinary interleukin-1 receptor antagonist/interleukin-1beta ratio indicate good prognosis in renal transplant recipients?

BACKGROUND: Interleukin-1 (IL-1) is produced by activated monocytes/macrophages; highly increased amounts of IL-1 have been found in renal tissue in acute rejection of renal grafts. The endogenous inhibitor of IL-1, interleukin-1 receptor antagonist (IL-1ra), is produced in many cells in response to the same stimulus as IL-1. There is some evidence that the balance between IL-1 and IL-1ra is important in the regulation of inflammatory responses. In many inflammatory diseases in both humans and animals, a high concentration of endogenous IL-1ra or administration of exogenous IL-1ra has been shown to relate to shorter recovery time or to reduced mortality. METHODS: We measured the urinary excretion of IL-1ra and IL-1beta during the first 3-6 posttransplant weeks in 23 patients with acute rejection (69 24-hr urine samples) and in 17 patients with stable graft function (51 24-hr urine samples) and expressed the results as cytokine/creatinine ratios. RESULTS: Within the follow-up time, patients with rejection had higher urinary IL-1beta/creatinine (ng/mmol) ratios (median 5.0 vs. 2.7; P<0.005), lower IL-1ra/creatinine (ng/mmol) ratios (median 18.1 vs. 34.2; P<0.1), and consequently lower IL-1ra/IL-1beta ratios (median 3.6 vs. 20.3, P<0.005), compared with patients without rejection. In rejecting patients, IL-1ra/creatinine was constantly low and decreased even further during acute rejection, whereas IL-1beta/creatinine ratios increased from a median prerejection value of 3.5 (range not measurable to 9.0) to a median value of 8.1 (P<0.0005) (range 1.6 to 18.3) during rejection. CONCLUSION: These results suggest that patients who produce high amounts of IL-1ra in relation to IL-1beta are less prone to acute allograft rejection than patients with low IL-1ra/IL-1beta ratios.     

Interleukin-1beta and the interleukin-1 receptor antagonist act in the striatum to modify excitotoxic brain damage in the rat

The cytokine interleukin-1 (IL-1) has been implicated in ischaemic, traumatic and excitotoxic brain damage. The results presented here reveal novel actions of IL-1 in the striatum which markedly exacerbate cortical neuronal damage elicited by local excitotoxins in the striatum or cortex. Intrastriatal infusion of IL-1 receptor antagonist, IL-1ra, markedly inhibited striatal neuronal damage caused by N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor activation in the rat. In contrast, intracortical infusion of IL-1ra failed to inhibit NMDA or AMPA receptor-induced damage in the cortex. Intrastriatal co-infusion of IL-1 with the NMDA or AMPA receptor agonist did not affect local striatal damage induced by activation of either glutamate receptor subtype, but caused extensive cortical damage when administered into the striatum with AMPA. This secondary damage was significantly reduced by pretreatment with the NMDA receptor antagonist (MK-801), which did not affect local (striatal) damage caused by AMPA. Infusion of IL-1beta into the striatum (but not the cortex) markedly enhanced cortical damage caused by infusion of an NMDA or AMPA receptor agonist into the cortex. These data reveal selective actions of IL-1 and IL-1ra in the striatum, which influence cortical neuronal loss and suggest that IL-1 selectively enhances damage caused by AMPA receptor activation.     

Agonist-induced phosphorylation of the luteinizing hormone/chorionic gonadotropin receptor expressed in a stably transfected cell line

Much of the definitive work on G-protein-coupled receptor phosphorylation and its impact on receptor function has been performed with the catecholamine receptors. Evidence for receptor phosphorylation is lacking, however, for G-protein-coupled receptors that bind larger ligands, such as LH/CG. Using immunoprecipitation techniques and a clonal cell line stably transfected with the LH/CG receptor, we show here for the first time that exposure of cells to hCG induces phosphorylation of its cognate receptor. The hCG-induced increase in receptor phosphorylation requires receptor activation because it cannot be elicited with a hCG antagonist and is mediated at least in part by the cAMP second messenger system. This hypothesis is supported by the finding that the hCG-induced receptor phosphorylation is greatly reduced (but not abolished) in a cell line that overexpresses cAMP phosphodiesterase and that receptor phosphorylation can be induced by activation of endogenous cAMP synthesis with prostaglandin E2 or by addition of 8-bromo-cAMP. Last, we show that LH/CG receptor phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. We also examined a potential correlation between LH/CG receptor phosphorylation and uncoupling of the receptor from its effector. Although the phorbol ester-induced phosphorylation of the LH/CG receptor can be correlated with uncoupling, other experiments indicate that hCG-induced uncoupling of the LH/CG receptor can occur under conditions where the cAMP-mediated receptor phosphorylation is greatly reduced (or abolished).