Long-term inhibition of dipeptidyl-peptidase 4 reduces islet infiltration and downregulates IL-1β and IL-12 in NOD mice

Dipeptidyl-peptidase 4 (DPP-4) inhibitor (sitagliptin) is a novel anti-hyperglycemia drug in the treatment of type 2 diabetes. However, its potential in type 1 diabetes is still unclear. Recent studies show that increased infection, especially respiratory tract infection, is significantly associated with DPP-4 inhibitors. In this study, we aimed to explore the effects of long-term inhibition of DPP- 4 on innate immunity in type 1 diabetes. Forty mice were randomly divided into 4 groups (n = 10 in each group): control group, lipopolysaccharide (LPS) group, sitagliptin group and sitagliptin + LPS group. The concentrations of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, TNF-α and IFN-γ were measured with Mesco Scale Discovery multiplexed-assay kit. Immunohistochemistry staining of pancreases was performed and insulitis scores for each islet were determined. The results showed that DPP-4 inhibition has no effect on incident rate of diabetes and metabolic parameters in NOD mice. Long-term inhibition of DPP-4 reduced CD4+T cells to infiltrate into islets and ameliorated insulitis in NOD mice. DPP-4 inhibition downregulated serum interleukin IL-1β and IL-12 in NOD mice. However, it had no significant effect on LPS-induced IL-1β, IL-6, IL-10, IL-12, tumor necrosis factor (TNF)-α and interferon (IFN)-γ in NOD mice. In conclusion, Long-term inhibition of DPP-4 exists anti-inflammatory effect in type 1 diabetes probably by reducing CD4+T cells to infiltrate into islets and downregulating L-1β and IL-12 in serum.     

Fasitibant prevents the bradykinin and interleukin 1β synergism on prostaglandin E2 release and cyclooxygenase 2 expression in human fibroblast-like synoviocytes

This study investigates the effect of the selective and potent B(2) receptor antagonist fasitibant (MEN16132) on the proinflammatory effect of bradykinin (BK) and its interaction with interleukin 1β (IL-1β) in human synoviocytes. PGE(2) content was detected in the surnatants and COX-2 and COX-1 gene and protein expression determined in the cells. Radioligand binding ([(3)?H]BK) and BK-induced inositolphosphate experiments were performed. Incubation of synoviocytes with BK induced a sustained production of PGE(2) and transient COX-2 gene expression that were prevented by pretreatment with fasitibant (1 μM, 30 min preincubation). IL-1β increased PGE(2) release and COX-2 expression more than BK alone. The combined treatment of cells with BK and IL-1β induced an even increase of released PGE(2) and COX-2 gene and protein expression indicating a synergistic rather than an additive effect, not related to an increase of B(2) receptors density or its coupling. These potentiating effects of BK on PGE(2) production and increased COX-2 expression produced by IL-1β were B(2)-receptor-mediated as fasitibant could prevent them. None of the treatments induced changes in the COX-1 expression. The synergistic PGE(2) production was abolished by the specific NF-kappaB inhibitor (BAY-117085), whereas specific inhibitors for the p38 (SB203580), JNK (SP600125), and ERK1/2 (PD98059) mitogen-activated protein kinases could prevent the prostanoid release. BK can potentiate the COX-2 gene expression and consequent prostanoid production induced by IL-1β. The prevention of this synergism by fasitibant indicates BK B(2) receptor blockade as an alternative symptomatic therapy for osteoarthritis.     

Interferon-γ reduces tumor-induced Ia− macrophage-mediated suppression: role of prostaglandin E2, Ia,and tumor necrosis factor-α

Tumor growth enhances macrophage (Mφ) suppressor activity by causing Mφ to increase synthesis of inhibitory molecules such as prostaglandin E₂ (PGE₂) or decreasing their expression of up-regulatory molecules such as the class II MHC protein Ia. Although these tumor-induced changes are correlated, it is unknownwhether tumor-bearing host (TBH) Ia⁻ Mφ become more suppressive by increasing their PGE₂ synthesis. To assess the role of PGE₂ in tumor-induced Ia⁻ Mφ-mediated suppression of CD4⁺ T-cell alloreactivity, unseparated (Ia⁺ -enriched) or Ia⁺ -depleted (Ia⁻) populations of murine normal host (NH) or TBH splenic Mφ were added to mixed lymphocyte reaction (MLR) cultures. NH or TBH Ia⁻ Mφ were significantly more suppressive than their respective unseparated populations, and TBH Ia⁻ Mφ were more suppressive than their NH counterparts. When PGE₂ production was blocked with indomethacin, TBH Ia⁻ Mφ-mediated suppression was reduced more than suppression mediated by all other Mφ populations. A PGE₂-specific ELISA showed more PGE₂ in Ia⁻ Mφ-containing cultures than in those with whole Mφ and more cultures containing TBH Ia⁻ Mφ than in their NH counterparts. Because interferon-γ (IFN-γ) is a potent Mφ activation molecule that regulates both Ia expression and PGE₂ production, the effects of IFN-γ on tumor-induced Ia⁻ Mφ-mediated suppression were investigated. Exogenous IFN-γ reduced suppression mediated by all Mφ populations except NH unseparated Mφ. IFN-γ suppressed alloreactivity without Mφ or with NH unseparated Mφ. Suppression mediated by NH or TBH Ia⁻, and TBH unseparated Mφ was also reduced when Mφ were pre-incubated with IFN-γ before their addition to MLR cultures. IFN-γ addition did not block Ia⁻ Mφ-mediated suppression by decreasing Mφ PGE₂ production. In fact, IFN-γ addition increased PGE₂ production two-fold in MLR cultures. However, IFN-γ partly reduced suppression mediated by exogenous PGE₂ added to Mφ-depleted cultures. Cytofluorometric analysis showed that IFN-γ increased the percentage of Ia⁺ Mφ in NH and TBH Ia⁻ Mφ populations. Blocking TNF-α activity with anti-TNF-α antibodies caused IFN-γ to suppress alloreactivity in all Mφ-added cultures. Collectively, these data show that tumor-induced suppression mediated by Ia⁻ Mφ is caused by increased PGE₂ synthesis. IFN-γ strongly reduces Ia⁻ Mφ-mediated suppression by blocking PGE₂-mediated suppression, enhancing Ia⁻ Mφ production of the up-regulatory molecule TNF-α, and possibly by increasing the number of Ia⁺ Mφ. These effects of IFN-γ on Ia⁻ Mφ suggest that this cytokine increases immunity and Mφ-mediated cytotoxicity during cancer.     

α1-Adrenoceptor subtype mediating contraction of the smooth muscle in the lower urinary tract and prostate of rabbits

Summary The -adrenoceptor subtype mediating contraction of the smooth muscle in the urinary bladder base (trigone), proximal urethra and prostate isolated from male rabbits was investigated by comparing the responsiveness to -adrenoceptor agonists and antagonists under condition where -adrenoceptors and neuronal and extraneuronal uptakes were inhibited. Noradrenaline (non-selective), phenylephrine (₁-selective) and clonidine (₂-selective) caused a dose-dependent contraction in the trigone, urethra and prostate. Phenylephrine acted as a full agonist whereas clonidine was a partial agonist. YM-12617 and prazosin (₁-selective), phentolamine (non-selective) and yohimbine (₂-selective) produced dose-dependent shifts to the right of the dose-response curves for noradrenaline, phenylephrine and clonidine in the all three tissues. YM-12617 (pA₂=9.77, 9.67 and 9.73 for trigone, urethra and prostate, respectively), prazosin (pA₂=8.26, 8.20 and 8.08), phentolamine (pA₂=7.67, 7.62 and 7.45) and yohimbine (pA₂=6.30, 6.30 and 5.94) showed constant pA₂ values irrespective of the agonists and tissues used, suggesting that only a single subclass of -adrenoceptors is present. The actual pA₂ values for these antagonists are comparable to those reported previously in tissues said to contain mainly ₁-adrenoceptors. Thus, we concluded that the postsynaptic -adrenoceptors of the rabbit trigone, urethra and prostate mediating contraction belong to the ₁-subtype.     

A comparison of the central actions of prostaglandins A1, E1, E2, F1α, and F2α in the rat

The effect of prostaglandins (PGs) A₁, E₁, E₂, F₁, and F₂ administered intraventricularly at doses of 0.02–4.0 g/rat were studied in some behavioral, antinociceptive and anticonvulsant tests in rats. Exploratory and locomotor activity were decreased by all PGs except A₁ and F₂ which had no effect on locomotor activity. All PGs studied, except A₁, induced hyperthermia and afforded protection in the hot-plate analgesic test and against maximal electroshock seizures.     

A comparison of the central actions of prostaglandins A1, E1, E2, F1α, and F2α in the rat

Prostaglandins (PGs) injected into the right lateral brain ventricle (i.v.c.) of the rat increased the sleeping time induced by hexobarbital, chloral hydrate, and ethanol. PGE₁ and PGE₂ intensified chlorpromazine-induced catalepsy, inhibited amphetamine hyperactivity, and significantly depressed the amphetamine-induced stereotypy. NA concentrations were decreased by PGE₁ and PGE₂ and were increased by PGF₂. PGF₂ increased both 5-HT and 5-HIAA levels in rat brain. Total ACh concentrations were increased by PGF₁ and PGF₂. PGE₁, PGE₂, and PGF₂ enhanced the turnover of NA, DA, and 5-HT. PGE₂ counteracted the decreased activity induced by -MT and abolished the hypothermic action of -MT. PGF₂ had little effect on the activity of PCPA pretreated rats, whereas the higher doses of PGF₂ increased body temperature in these animals.     

NF-κB-dependent IL-8 induction by prostaglandin E2 receptors EP1 and EP4

Background and Purpose

Recent studies suggested a role for PGE₂ in the expression of the chemokine IL-8. PGE₂ signals via four different GPCRs, EP₁-EP₄. The role of EP₁ and EP₄ receptors for IL-8 induction was studied in HEK293 cells, overexpressing EP₁ (HEK-EP₁), EP₄ (HEK-EP₄) or both receptors (HEK-EP₁ + EP₄).

Experimental Approach

IL-8 mRNA and protein induction and IL-8 promoter and NF-κB activation were assessed in EP expressing HEK cells.

Key Results

In HEK-EP₁ and HEK-EP₁ + EP₄ but not HEK or HEK-EP₄ cells, PGE₂ activated the IL-8 promoter and induced IL-8 mRNA and protein synthesis. Stimulation of HEK-EP₁ + EP₄ cells with an EP₁-specific agonist activated IL-8 promoter and induced IL-8 mRNA and protein, whereas a specific EP₄ agonist neither activated the IL-8 promoter nor induced IL-8 mRNA and protein synthesis. Simultaneous stimulation of HEK- EP₁ + EP₄ cells with both agonists activated IL-8 promoter and induced IL-8 mRNA to the same extent as PGE₂. In HEK-EP₁ + EP₄ cells, PGE₂-mediated IL-8 promoter activation and IL-8 mRNA induction were blunted by inhibition of IκB kinase. PGE₂ activated NF-κB in HEK-EP₁, HEK-EP₄ and HEK-EP₁ + EP₄ cells. In HEK-EP₁ + EP₄ cells, simultaneous activation of both receptors was needed for maximal PGE₂-induced NF-κB activation. PGE₂-stimulated NF-κB activation by EP₁ was blocked by inhibitors of PLC, calcium-signalling and Src-kinase, whereas that induced by EP₄ was only blunted by Src-kinase inhibition.

Conclusions and Implications

These findings suggest that PGE₂-mediated NF-κB activation by simultaneous stimulation of EP₁ and EP₄ receptors induces maximal IL-8 promoter activation and IL-8 mRNA and protein induction.     

A potent PGK1 antagonist reveals PGK1 regulates the production of IL-1β and IL-6

Glycolytic metabolism enzymes have been implicated in the immunometabolism field through changes in metabolic status. PGK1 is a catalytic enzyme in the glycolytic pathway. Here, we set up a high-throughput screen platform to identify PGK1 inhibitors. DC-PGKI is an ATP-competitive inhibitor of PGK1 with an affinity of K_d = 99.08 nmol/L. DC-PGKI stabilizes PGK1 in vitro and in vivo, and suppresses both glycolytic activity and the kinase function of PGK1. In addition, DC-PGKI unveils that PGK1 regulates production of IL-1β and IL-6 in LPS-stimulated macrophages. Mechanistically, inhibition of PGK1 with DC-PGKI results in NRF2 (nuclear factor-erythroid factor 2-related factor 2, NFE2L2) accumulation, then NRF2 translocates to the nucleus and binds to the proximity region of Il-1β and Il-6 genes, and inhibits LPS-induced expression of these genes. DC-PGKI ameliorates colitis in the dextran sulfate sodium (DSS)-induced colitis mouse model. These data support PGK1 as a regulator of macrophages and suggest potential utility of PGK1 inhibitors in the treatment of inflammatory bowel disease.     

α1A-Adrenoceptor mediated contraction of rat prostatic vas deferens and the involvement of ryanodine stores and Ca2+ influx stimulated by diacylglycerol and PKC

1. The present study has investigated the α₁-adrenoceptor subtype mediating contraction of the rat isolated prostatic vas deferens and the possible effector mechanisms involved in this response by use of functional experiments.
2. Contractions to noradrenaline in the rat isolated prostatic vas deferens were antagonized by prazosin (9.4, 1.04±0.19, pA₂ and Schild plot slope), 5-methyl urapidil (8.9, 1.10±0.13), BMY 7378 (6.4, 1.53±0.07) and RS 17053 (8.3, 1.13±0.18). These affinities are consistent with the response being mediated by the α_1A-adrenoceptor subtype.
3. The contraction to noradrenaline at 37°C consisted of an initial phasic response, composed of many rhythmic contractile spikes and a more slowly developing tonic contraction. When the temperature was lowered to 25°C the phasic contraction became a smooth single response which was increased in magnitude.
4. In Ca²⁺-free Krebs solution the tonic contraction to noradrenaline (10⁻⁴ M) was abolished, suggesting that this response was dependent on influx of extracellular Ca²⁺. After 2 min in Ca²⁺-free Krebs solution at 37°C and 25°C the phasic response to noradrenaline (10⁻⁴ M) was 38±2% and 91±4%, respectively, compared with the phasic contraction to noradrenaline (10⁻⁴ M in normal Krebs solution) and after 30 min it was abolished at 37°C and was 7±1% at 25°C. Ryanodine abolished the noradrenaline response in Ca²⁺-free Krebs solution for 2 min at 25°C, while cyclopiazonic acid reduced it to 36±2%.
5. In normal Krebs solution at 25°C the protein kinase C inhibitor calphostin C reduced the tonic contraction to noradrenaline (10⁻⁵ M) from 36±8% to 14±3% compared with the phasic contraction to noradrenaline (10⁻⁴ M). The DAG kinase inhibitor R 59022 increased the contraction following the initial phasic response to a maximum of 107±17% after 35 s, before dropping down to a well maintained contraction which was still greater in magnitude compared with the control. Nifedipine (3×10⁻⁷ M) reduced the tonic contraction from 49±6% to 7±1% but did not reduce the phasic response. Ryanodine (10⁻⁴ M) reduced the phasic contraction from 50±2% to 7±1% and the tonic response from 47±5% to 27±5%.
6. The phorbol ester phorbol-12,13-dibutyrate at 25°C produced a transient contraction of the rat prostatic vas deferens, maximum response (10⁻⁵ M) 48±4%, compared with the maximum tonic response to noradrenaline. The contraction to PDBu (10⁻⁵ M) was reduced to 23±2% by calphostin C (10⁻⁶ M) and to 15±1% by nifedipine (3×10⁻⁷ M) and was abolished after 2 min in Ca²⁺-free Krebs solution.
7. In conclusion, the α_1A-adrenoceptor mediated contraction to noradrenaline of the rat prostatic vas deferens appears to consist of an initial phasic component due to the release of intracellular Ca²⁺ from ryanodine-sensitive stores. These stores are depleted in the absence of extracellular Ca²⁺ and this depletion is slower at 25°C than at 37°C. The phasic contraction is followed by a tonic contraction involving activation of protein kinase C by diacylglycerol and influx of Ca²⁺ through nifedipine-sensitive channels.

     

Combined exercise training reduces IFN-γ and IL-17 levels in the plasma and the supernatant of peripheral blood mononuclear cells in women with multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disorder in which lymphocytic infiltration mediated mainly by pro-inflammatory cytokines. In this study, we examined the effect of combined exercise training on the levels of IFN-γ, IL-4 and IL-17 in the plasma and the supernatant of peripheral blood lymphocytes in women with multiple sclerosis. Expanded Disability Status Scale (EDSS), VO₂max, muscle strength, and balance tests were obtained at baseline and post-treatment follow-up. Combined exercises training was designed for 24 sessions during 8 weeks. Each session was started with 5 min warm-up and was followed by 10 min stretch training, 20 min aerobic exercises and 20 min resistance–endurance training. The disability score was significantly decreased in test MS subjects after 8 weeks combined exercise training. Muscle strength and balance were increased significantly after the training program in test group. In this study, plasma, and peripheral blood mononuclear cell (PBMC) IL-17 and IFN-γ production was significantly decreased after 8 weeks combined training. Our findings suggest that combined training has useful anti-inflammatory effects by decrease in PBMC and plasma IL-17 production.     

Interleukin-1β (IL-1β) increases pain behavior and the blood glucose level: possible involvement of sympathetic nervous system

The relationship between interleukin-1β (IL‐1β)‐induced nociception and the blood glucose level was studied in ICR mice. We found in the present study that intrathecal (i.t.) injection of IL‐1β increased pain behavior. In addition, i.t. IL‐1β injection caused an elevation of the blood glucose level. The time‐course study showed that maximal blood glucose level was observed 30 and 60 min after i.t. IL‐1β administration. Furthermore, i.t. injection of IL‐1β enhanced the blood glucose level when mice were orally fed with d‐glucose. The i.t. administration of IL‐1β antagonist (AF12198) inhibited the hyperglycemia and pain behaviors induced by IL‐1β. We found in the present study that adrenal tyrosine hydroxylase (TH) mRNA level was also increased by i.t. IL‐1β injection. Furthermore, intraperitoneal (i.p.) pretreatment with phentolamine (an α₁‐adrenergic blocker) or yohimbine (an α₂‐adrenergic blocker) significantly attenuated the blood glucose level and pain behavior induced by IL‐1β administered i.t. However, the blood glucose level and pain behavior were not affected by butoxamine (a β₂-adrenergic blocker), whereas metoprolol (a β₂-adrenergic blocker) enhanced IL‐1β-induced blood glucose level and pain behavior in mice fed with d‐glucose. However, its effect was not statistically significant. Our results suggest that IL‐1β administered i.t. increases the blood glucose level via an activation of α adrenergic nervous system.     

IL-1α and IL-1β as alternative biomarkers for risk assessment and the prediction of skin sensitization potency

Potential biomarkers of skin sensitization in RAW264.7 mouse macrophages were investigated as alternatives to animal experiments and risk assessment. The concentrations that resulted in a cell viability of 90% (CV90) and 75% (CV75) were calculated by using a water-soluble tetrazolium salt (WST)-1 assay and used to analyze the skin sensitization potency of 23 experimental materials under equivalent treatment conditions. In addition, the expression of interleukin (IL)-1α, IL-1β, IL-31, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2), and cyclooxygenase-2 (COX-2) was analyzed utilizing Western blotting. In the cell viability analysis, skin sensitizers were generally more cytotoxic and exhibited increased skin sensitization potency. However, nonsensitizers did not show any marked cytotoxic tendency. Biomarker analysis demonstrated that IL-1α, IL-1β, and the combination of IL-1α and IL-1β (IL-1α + IL-1β) predicted reliably skin sensitization potential (1) sensitivities of 94.4%, 83.3%, and 83.3%, specificities of 100%, 100%, and 100%, and (2) accuracies of 95.7%, 87%, and 87%, respectively. These observations correlated most reliably as indicators for skin sensitization potency. Data suggest that IL-1α and IL-1β may serve as potential biomarkers for skin sensitization and provide an alternative method to animal experiments for prediction of skin sensitization potency and risk assessment.     

Involvement of interleukin-1β, nerve growth factor and prostaglandin E2 in endotoxin-induced localized inflammatory hyperalgesia

1. Intraplantar endotoxin (ET) injection (1.25 μg) into the hind paw of rats resulted in a localized inflammatory hyperalgesia, as assessed by paw pressure (PP), paw immersion (PI), tail flick (TF) and hot plate (HP) tests.
2. ET injection resulted in a significant elevation in the levels of interleukin-1β (IL-1β) and nerve growth factor (NGF) in the injected foot as compared with the non-injected foot. This increase was attenuated by intraperitoneal injections of dexamethasone (200 and 400 μg kg⁻¹) and to a lesser extent by indomethacin (2 and 8 mg kg⁻¹).
3. The tripeptide Lys-D-Pro-Val, which is known to antagonize IL-1β and prostaglandin E₂ (PGE₂) reversed mechanical hyperalgesia, as assessed by the PP test, and reduced significantly thermal hyperalgesia, as assessed by the HP and TF tests.
4. IL-1ra reversed both mechanical (PP) and thermal (PI) nociceptive thresholds tested on the injected leg and significantly reduced thermal hyperalgesia, as assessed by the HP and TF tests.
5. A sheep, anti-mouse NGF antiserum reversed mechanical hyperalgesia (PP test) but had little or no effect on thermal hyperalgesia (PI, HP and TF tests).
6. Our results indicate the importance of IL-1β, NGF and prostaglandin E₂ (PGE₂) in the development of ET induced hyperalgesia and the possible existence of different mechanisms underlying thermal and mechanical as well as central and peripheral hyperalgesia.

     

IL-1β enhances vascular smooth muscle cell proliferation and migration via P2Y2 receptor-mediated RAGE expression and HMGB1 release

Vascular smooth muscle cells (VSMCs) are the major cell type in blood vessel walls, and their proliferation and migration play important roles in the development of atherosclerosis. Recently, it has been reported that IL-1β mediates the inflammatory response through the upregulation of the P2Y₂ receptor (P2Y₂R). Thus, we examined the role of P2Y₂R in IL-1β-mediated proliferation and migration of VSMCs and the underlying molecular mechanisms. VSMCs were pretreated with IL-1β for 24 h to upregulate P2Y₂R expression. The cells were then stimulated with UTP or ATP for the indicated times, and cell proliferation and migration and the related signaling pathways were examined. The equipotent P2Y₂R agonists ATP and UTP enhanced proliferation, RAGE expression and HMGB1 secretion in IL-1β-pretreated VSMCs. Additionally, pretreatment with IL-1β enhanced UTP-mediated VSMC migration and MMP-2 release, but these effects were not observed in the P2Y₂R-siRNA- or RAGE-siRNA-transfected VSMCs. Next, the signaling molecules involved in P2Y₂R-mediated cell proliferation and migration were determined. The ERK, AKT, PKC, Rac-1 and ROCK2 pathways were involved in UTP-induced cell proliferation and migration, MMP-2 and HMGB1 secretion and RAGE expression in the IL-1β-pretreated VSMCs. UTP induced the phosphorylation of ERK, AKT and PKC and the translocation of Rac-1 and ROCK2 from cytosol to membrane as well as stress fiber formation, which were markedly increased in the IL-1β-pretreated VSMCs but not in the P2Y₂R-siRNA-transfected VSMCs. These results demonstrate that pro-inflammatory cytokines associated with atherosclerosis, such as IL-1β, can accelerate the process of atherosclerosis through the upregulation of P2Y₂R.     

Effects of tyrosine kinase inhibitor,genistein, and phosphotyrosine—phosphatase inhibitor,orthovanadate, on Ca2+-free contraction of uterine smooth muscle of the rat

• 1.1. The effects of a protein-tyrosine kinase inhibitor, genistein, and a protein-tyrosine phosphatase inhibitor, orthovanadate, were tested on the Ca²⁺-free contraction of the estrogen-dominated rat, which has been proved to be induced mainly via protein kinase C entirely independently of Ca²⁺.
• 2.2. Genistein (30 μM) significantly inhibited the contraction indicating participation of tyrosine kinase activity in the contraction.
• 3.3. Orthovanadate caused contraction concentration-dependently and augmented the Ca²⁺-free contraction at concentrations of more than 1 μM. The contraction by orthovanadate was not inhibited so significantly by genistein (30 μM).
• 4.4. Possible participation of tyrosine kinase activity in Ca²⁺-free contraction is discussed in addition to the formerly reported participation of protein kinase C.

     

Expression and function of β-arrestin 2 stimulated by IL-1β in human fibroblast-like synoviocytes and the effect of paeoniflorin

The aim of this study was to explore the expression and function of β-arrestin 2 in human fibroblast-like synoviocytes (FLS) stimulated by IL-1β and the effect of paeoniflorin (Pae). We isolated and cultured human FLS, which were stimulated by IL-1β. The FLS proliferations were detected by [3H] thymidine incorporation. The level of cAMP stimulated by IL-1β on different times was investigated by radioimmunoassay, and the activity of PKA was measured by luminescent kinase assay. The expression of β-arrestin 2 was measured by western blot. We found that the human FLS proliferation increased apparently in 24 h, and the activities of PKA and cAMP accumulation increased significantly in 6 h after stimulated by IL-1β, while cAMP accumulation and the activities of PKA decreased especially in 24 h when the expression of β-arrestin 2 increased significantly. Decreased cAMP accumulation and the increased expression of β-arrestin 2 may reveal a positive correlation with the FLS proliferation. Pae (10^- 5, 10^- 6, 10-7 mol?L^- 1) in vitro could suppress the FLS proliferation and the high expression of β-arrestin 2. The expression of β-arrestin 2 may have a positive correlation with the human FLS proliferation, while the activities of PKA and cAMP accumulation have a negative correlation with the proliferation. The increased β-arrestin 2 may down-regulate cAMP-PKA signaling pathway and promote FLS proliferation. Pae may suppress the expression of β-arrestin 2 and up-regulate cAMP-PKA signaling, which may be one of the mechanisms for the effects of Pae on inhibiting human FLS proliferation.     

The role of α1-adrenoceptor subtypes in the phasic and tonic responses to phenylephrine in the longitudinal smooth muscle of the rat portal vein

Summary The purpose of this investigation was to determine whether ₁-adrenoceptor subtypes (co)exist in the rat portal vein and, if so, whether they could be functionally associated with the phasic and tonic types of contraction as a response to ₁-adrenoceptor stimulation by phenylephrine.A low Ca²⁺ concentration (0.9 mmol/l) in the Tyrode solution enabled us to quantify changes both in the phasic myogenic activity and in the basal tone of the rat portal vein preparation very precisely. We used both competitive and non-competitive -adrenoceptor antagonists which have been employed successfully by other investigators to discriminate between ₁-adrenoceptor subtypes in vascular and other tissues. Schild analysis showed that the competitive -adrenoceptor antagonists prazosin, phentolamine, yohimbine, corynanthine, idazoxan, rauwolscine and 5-methyl-urapidil could not distinguish between the phasic and tonic responses to phenylephrine and/or different ₁-adrenoceptor subtypes in the rat portal vein. However, when we compared our pA₂ values with those found to be representative indicators according to subclassifications based on the use of selective antagonists in different tissues, the ₁-adrenoceptors in the rat portal vein appeared to belong to the _1L- or _1a-subtype. This subclassification was not in accordance with the data obtained with the irreversible -cadrenoceptor antagonist chloroethylclonidine. However, the validity of this alkylating agent as a tool for receptor classification was restricted, at least in the rat portal vein, by its effects on receptor reserve. In contrast to the competitive -adrenoceptor antagonists, the irreversible -adrenoceptor antagonists phenoxybenzamine and chloroethylclonidine could indeed discriminate between the phasic and tonic types of contraction in response to ₁-adrenoceptor stimulation by phenylephrine, indicating two different receptor reserves for phenylephrine for the two types of responses.In conclusion, both the phasic and tonic types of contraction elicited by phenylephrine in the longitudinal smooth muscle of the rat portal vein appear to be mediated by one particular ₁-adrenoceptor subtype as defined by Schild analysis with selective, competitive -adrenoceptor antagonists. However, using the method of receptor alkylation with phenoxybenzamine, two different affinity constants for the two types of responses could be calculated for phenylephrine. This may reflect the involvement of two different subtypes of ₁-adrenoceptors or more probably, the existence of only one ₁-adrenoceptor subtype, which is coupled with two different intrinsic efficacies to the effector pathways mediating the phasic and tonic responses, respectively. Send offprint requests to H. R. Schwietert at the above address     

Action of prostaglandin,PGF2α, on the uterus of the pregnant rat

Summary The effects of prostaglandin F₂ (PG) have been studied on the transmembrane potentials and contractions in isolated myometrial strips from pregnant rats. The results showed that: 1. The sensitivity of the myometrium to exogenous PG increases from day 19 to day 22 of gestation. 2. The electrical response to PG, at maximally effective doses (10^–6 to 10^–5 M) consists of a slow depolarization which upon reaching threshold initiates spike discharge. 3. These actions are most pronounced t term (day 22) and are due to a direct action of PG on the myometrical cells. 4. D-600 (a methoxy derivative of verapamil) abolishes spike discharge and the phasic contractions induced by PG but has no effect on the slow depolarization and the accompanying increase in tonic tension. 5. The slow depolarization is dependent upon the presence of sodium in the external environment and is unaffected by the removal of calcium. 6. The spikes (and phasic contractions) are dependent upon the presence of calcium in the external environment.This study was supported by USPHS Grant HD-06963-9