Effects of oxymatrine on the serum levels of T helper cell 1 and 2 cytokines and the expression of the S gene in hepatitis B virus S gene transgenic mice: a study on the anti-hepatitis B virus mechanism of oxymatrine

BACKGROUND: Oxymatrine has been shown to have a remarkable inhibitory activity to hepatitis B virus (HBV) infection with a hepatitis B virus e antigen (HBeAg) serum conversion rate of approximately 45%. In order to explore the anti-HBV mechanism of oxymatrine, the effects of oxymatrine on serum levels of T helper (h)1 cytokines (interferon (IFN)-gamma and interleukin (IL)-2) and Th2 cytokines (IL-4 and IL-10), and the expression of S gene in HBV S gene transgenic mice were studied. METHODS: Each transgenic mouse was either injected with oxymatrine or saline intraperitoneally once a day for 30 days. Serum levels of IFN-gamma, IL-2, IL-4 and IL-10 were quantitated and compared to the data before the treatment. The expression of HBV S gene in transgenic mice was analyzed at the DNA, mRNA and protein levels. RESULTS: The serum levels of IFN-gamma in transgenic mice before or after oxymatrine treatment were 3.108 +/- 3.172 and 11.059 +/- 6.971 pg/mL, respectively. In contrast, serum levels before and after oxymatrine treatment for IL-4 were 29.045 +/- 13.235 and 13.024 +/- 9.002 pg/mL, respectively (P < 0.001). The serum levels of IL-2 in the control (saline injection) and oxymatrine-treated mice were 1.070 +/- 0.447 and 5.537 +/- 2.887 pg/mL, respectively (P < 0.0001); and that of IL-10 were 97.226 +/- 73.306 and 33.607 +/- 23.154 pg/mL, respectively (P < 0.01). No significant differences were observed in the expression of HBV S gene in the transgenic mice at the DNA, mRNA and protein levels before or after oxymatrine treatment. CONCLUSIONS: The fact that Th1 cytokines are increased while Th2 cytokines are decreased suggests that oxymatrine treatment triggers the change of immune response to hepatitis B infection in transgenic mice, which leads to improved HBV inhibitory activities. The study can help us better understand the mechanisms of the anti-HBV drug, oxymatrine, and how it has potential as an application in clinical chronic hepatitis B treatment.     

Modulation of the beta-adrenergic receptor system of vascular smooth muscle cells in vitro and in vivo by chronically elevated endothelin-1 levels

Endothelin-1 (ET-1) levels are chronically elevated in several cardiovascular diseases and correlate with an increased mortality. However, in contrast to acute biological activities such as vasoconstriction, little is known about long-term effects of ET-1. In this study we determined the effects of ET-1 on the beta(2)-adrenergic receptor (AR) system. Incubation of smooth muscle cells with ET-1 for 72 hr led to increased beta(2)AR density as determined by radioligand binding. Experiments with inhibitors of protein and RNA synthesis as well as RT-PCR revealed that beta(2)AR upregulation required de novo synthesis. In addition, protein kinase C but neither NO nor prostaglandin metabolism were involved in this effect. The enhanced expression of beta(2)AR was associated with an increased expression of its stimulatory G-protein and the receptor's ability to stimulate adenylyl cyclase. To study chronic effects of ET-1 in vivo, rats were infused with ET-1 for 3 weeks. Similarly as in cultured cells, prolonged ET-1 exposure led to increased betaAR expression in vivo. As a consequence, beta(2)AR-induced vasodilatation was increased in aortic rings from ET-1-treated animals. Our results therefore suggest that chronically elevated ET-1 levels in vitro and in vivo induce counterregulatory mechanisms by increasing betaARs that attenuate the vasoconstrictive effects of ET-1.     

Decreased G-protein coupling of serotonin 5-HT(1A) receptors in the brain of 5-HT(1B) knockout mouse

The firing of central serotonin (5-hydroxytryptamine, 5-HT) neurons and their capacity to release 5-HT are subjected to a receptor-mediated auto-control via 5-HT(1A) and 5-HT(1B) receptors respectively located on the somata/dendrites (5-HT(1A) autoreceptors) and preterminal axon arborizations (5-HT(1B) autoreceptors) of these neurons. To further characterize mutual adaptations of these two receptor subtypes in the absence of one of them, activation of G-protein coupling by agonist was measured and compared to wild-type (WT) in 5-HT(1A) and 5-HT(1B) homozygous knockout (KO) mice. As expected, in WT, the non-selective 5-HT(1A/1B) receptor agonist 5-carboxyamidotryptamine (5-CT) stimulated guanosine 5'-O-(gamma-[(35)S]thio)triphosphate ([(35)S]GTP(gamma)S) incorporation in many brain regions endowed with one and/or the other receptor. In the respective KOs, no stimulation was measured in regions known to express only or mainly the deleted receptor. In the 5-HT(1A) KOs, the amplitude of G-protein activation in regions endowed with 5-HT(1B) receptors was unchanged by comparison to WT. In the 5-HT(1B) KOs, the magnitude of the 5-CT stimulation was the same as WT in all regions containing 5-HT(1A) receptors, except in the amygdala, where it was significantly lower, even if this region was one of the most strongly activated in the WT. A similar result was obtained in the amygdala of 5-HT(1B) KOs after activation by the selective 5-HT(1A) receptor agonist R-(+)8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT). Under these conditions, however, there was in addition a significant lowering of the stimulated (but not basal) [(35)S]GTP(gamma)S incorporation by comparison to WT in all regions endowed with 5-HT(1A) receptors, including the dorsal raphe nucleus. Thus, eventhough agonist radioligand binding to either 5-HT(1A) or 5-HT(1B) receptors is unchanged in the reciprocal KOs, it appears that a compensatory decrease in the efficiency of G-protein coupling to 5-HT(1A) receptors has developed in the 5-HT(1B) mutant. This could represent the first indication of a cross-talk between these two 5-HT receptor subtypes, at least in brain regions where they are co localized in the same neurons.     

The influence of charge clustering on the anti-HIV-1 activity and in vivo distribution of negatively charged albumins

The substitution of human serum albumin with negatively charged molecules, such as succinic acid (Suc-HSA) or aconitic acid (Aco-HSA), resulted in proteins with potent anti-HIV activities, by binding to viral gp120 (V3 loop). The aim of the present study was to investigate whether the distribution of negative charges on the albumin backbone influences the anti-HIV activity. Therefore, we prepared albumins with clusters of negatively charged groups by coupling of heparin. The effects of this substitution on anti-HIV activity, in vivo distribution and the protein structure as compared to random succinylation were assessed. In vitro studies indicated that HSA-modified with heparin 6 or 13 kD displayed anti-HIV activity (IC50=660 and 37 nM, respectively) and exhibited affinity for gp120-V3 loop, although the activity was lower than that of Suc-HSA. Combined derivatization of HSA with heparin 13 kD and aconitic acid groups resulted in significantly increased inhibitory actions (IC50=2.8 nM). Structural analysis showed that modification of HSA with heparin did not lead to extensive unfolding of the protein, meaning that these modified proteins were still globular in structure. In contrast, succinylation of HSA resulted in a highly randomly coiled conformation. Dynamic light scattering experiments revealed that, at neutral pH, the heparin fragments attached to the protein were wrapped around the molecule rather than sticking out into the solution. In conclusion, coupling of sufficient clustered negative charges, by coupling of Hep-fragments, on HSA resulted in a clear anti-HIV activity of the protein. Yet, random distribution of anionic groups in the albumin seemed more optimal for in vitro anti-HIV activity. The higher plasma and lymphatic concentrations of Hep-HSA compared to Suc-HSA seemed more favorable for an anti-HIV activity in vivo.     

G protein-coupled receptor for nicotinic acid in mouse macrophages

The use of the HDL-elevating drug nicotinic acid in the treatment and prevention of atherosclerotic disease is limited by the frequent induction of skin flushing. The therapeutic effects of nicotinic acid are attributed to inhibition of lipolysis in adipose tissue via a G protein-coupled receptor, whereas the mechanism of flush induction by release of prostaglandin D(2) from macrophages is not understood. In this study, we investigated if macrophages contain nicotinic acid receptors. Specific guanine nucleotide sensitive binding sites for [(3)H]nicotinic acid were detected in membranes from mouse RAW 264.7 macrophages. Nicotinic acid and related heterocycles stimulated activation of pertussis toxin-sensitive G proteins. The rank orders of potency in macrophage membranes were identical for inhibition of [(3)H]nicotinic acid binding and G protein activation, and were pharmacologically indistinguishable from that of the G protein-coupled nicotinic acid receptor in spleen membranes. These results indicate that the effects of nicotinic acid on macrophages, spleen and probably adipocytes are mediated via an identical, unique G protein-coupled receptor.     

Exendin-4 increases insulin sensitivity via a PI-3-kinase-dependent mechanism: contrasting effects of GLP-1

The insulinotropic agent, exendin-4, is a long-acting analogue of glucagon-like peptide-1 (GLP-1) which improves glucose tolerance in humans and animals with diabetes, but the underlying mechanisms and the effects of exendin-4 on peripheral (muscle/fat) insulin action are unclear. Previous in vivo and clinical studies have been difficult to interpret because of complex, simultaneous changes in insulin and glucagon levels and possible effects on hepatic metabolism. Thus, the comparative effects of exendin-4 and GLP-1 on insulin-stimulated 2-[3H]deoxyglucose (2-DOG) uptake were measured in fully differentiated L6 myotubes and 3T3-adipocytes, including co-incubation with inhibitors of the PI-3-kinase (wortmannin) and mitogen-activated protein (MAP) kinase (PD098059) pathways. In L6 myotubes, there was a concentration-dependent and PI-3-kinase-dependent increase in insulin-stimulated 2-DOG uptake with exendin-4 and GLP-1, e.g. for exendin-4 the C(I-200) value (concentration of insulin required to increase 2-DOG uptake 2-fold) decreased from 1.3 +/- 1.4 x 10(-7)M (insulin alone, n=16) to 5.9 +/- 1.3 x 10(-8)M (insulin+exendin-4 0.1nM, n=18, P<0.03). A similar insulin-sensitizing effect was observed with exendin-4 in 3T3-adipocytes, but GLP-1 had no effect on adipocyte insulin sensitivity. In conclusion, this is the first direct evidence showing that exendin-4 increases insulin-stimulated glucose uptake in muscle and fat derived cells via a pathway that involves PI-3-kinase activation. Furthermore, the contrasting responses of exendin and GLP-1 in 3T3-adipocytes suggest that the peripheral insulin-sensitizing effect of exendin-4 (in contrast to the insulinotropic effect) does not involve the GLP-1 receptor pathway.     

Pharmacological characterisation of the goldfish somatostatin sst5 receptor

Somatostatin (somatotropin release inhibiting factor, SRIF), exerts its effects via specific G protein coupled receptors of which five subtypes have been cloned (sst1-5). Recently, SRIF receptors have also been cloned from fish tissues. In this study, goldfish sst5 receptors (gfsst5) were expressed and characterised in the Chinese hamster lung fibroblast cell line, that harbours the luciferase reporter gene driven by the serum responsive element (CCL39-SRE-Luci). The agonist radioligands [125I]-LTT-SRIF-28 ([Leu8, DTrp22, 125I-Tyr25]SRIF-28) and [125I][Tyr10]cortistatin-14 labelled similar receptor densities with high affinity and in a saturable manner (pKd: 9.99-9.71; Bmax: 300-350 fmol mg-1). 5'-Guanylyl-imidodiphosphate inhibited radioligand binding to some degree (38.5-57.9%). In competition binding studies, the pharmacological profile of SRIF binding sites defined with [125I]LTT-SRIF-28 and [125I][Tyr10]cortistatin-14 correlated significantly (r2=0.97, n=20). Pharmacological profiles of human and mouse sst5 receptors expressed in CCL39 cells correlated markedly less with those of the gfsst5 profile (r2=0.52-0.78, n > or = b16). Functional expression of the gfsst5 receptor was examined by measurement of agonist-induced luciferase expression and stimulation of [35S]GTPgammaS ([35S]guanosine 5'-O-(3-thiotriphosphate) binding. Profiles were similar to those achieved in radioligand binding studies (r2=0.81-0.93, n=20), although relative potency (pEC50) was reduced compared to pKd values. Relative efficacy profiles of luciferase expression and [35S]GTPgammaS binding, were rather divergent (r2=0.48, n=20) with peptides showing full agonism at one pathway and absence of agonism at the other. BIM 23056 (D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-D-Nal-NH2) acted as an antagonist on the effects of SRIF-14 (pKB=6.74 +/- 0.23) on stimulation of [35S]GTPgammaS binding. Pertussis toxin abolished the effect of SRIF-14 on luciferase expression and [35S]GTPgammaS binding suggesting coupling of the receptor to G(i)/G(o) proteins. In summary, the present studies demonstrate that the gfsst5 receptor has a similar pharmacological profile and transductional properties to mammalian sst5 receptors. The difference in efficacy profiles defined using different functional assays suggests numerous, agonist specific, conformational receptor states, and/or ligand-dependent receptor trafficking.     

Quantitative stoichiometry of G-proteins activated by mu-opioid receptors in postmortem human brain

Paradoxically, the potencies (EC(50)) of agonists stimulating [35S]GTPgammaS binding are several orders of magnitude lower than their affinities in receptor binding assays. We have investigated the quantitative stoichiometry of mu-opioid receptor-G-protein coupling in postmortem human brain. [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) displaced [3H]naloxone binding in a biphasic pattern. The ratio between K(i-low) and EC(50) of DAMGO stimulating [35S]GTPgammaS binding was lower than one. The K(A) of DAMGO was calculated following mu-opioid receptor alkylation by beta-funaltrexamine from [35S]GTPgammaS binding data using the "nested hyperbolic method", yielding K(A)/EC(50)>1. Thus, only 1.2 +/- 0.2% of mu-opioid receptors was needed to be occupied to achieve the half-maximal effect of DAMGO. The estimated ratio between the G-proteins activated by 10 microM DAMGO (determined by isotopic dilution curves) and the occupied-mu-opioid receptors was 1304. In conclusion, we have determined the stoichiometric and the kinetic parameters in the mu-opioid receptor-G-protein system.     

A novel compound RS-0466 reverses beta-amyloid-induced cytotoxicity through the Akt signaling pathway in vitro

In order to determine whether 5-[bis(carboxymethyl) amino]-2-carboxy4-cyano-3-thiopheneacetic acid distrontium salt (S12911-2) inhibits bone resorption by acting on the differentiation and/or function of osteoclasts, its effects were assessed on the 1,25-dihydroxyvitamin D(3)-induced expression of carbonic anhydrase II and vitronectin receptor in chicken bone marrow cells, and on the resorbing activity of authentic rat osteoclasts cultured on bone slices. S12911-2 dose-dependently inhibited, after a 6-day exposure, the expression of carbonic anhydrase II and vitronectin receptor in stimulated osteoclasts (46% and 40%, respectively, at 10(-3) M Sr(2+), P<0.05). A pre-incubation of bone slices with S12911-2 induced a dose-dependent inhibition of bone resorbing activity from 32% at 10(-4) M Sr(2+) to 66% at 10(-3) M Sr(2+) (P<0.05 in each case). A continuous incubation (10(-3) M Sr(2+)) induced a greater inhibition of bone resorbing activity (73%, P<0.05). The inhibition of bone resorption obtained specifically with S12911-2 is related to an inhibition of the differentiation and resorbing activity of the osteoclasts.     

Cloning and characterization of a novel human 5-HT4 receptor variant that lacks the alternatively spliced carboxy terminal exon. RT-PCR distribution in human brain and periphery of multiple 5-HT4 receptor variants.

We have cloned a novel C-terminal splice variant of serotonin 5-HT4 receptors from human hippocampus. The deduced protein extends only one aminoacid past the splicing point. We propose to call the novel variant h5-HT4(n) since it contains none of the C-terminal exons alternatively spliced in other variants. The pharmacological profile of h5-HT4(n) stably expressed in HeLa cells is in agreement with other reported variants. Stably transfected cells showed increased basal levels of intracellular cAMP in absence of agonist, indicating constitutive activity of the expressed receptors. 5-HT induced robust increases of intracellular cAMP. The 5-HT4 receptor antagonist GR 113808 blocked the effects of 5-HT and brought intracellular cAMP below basal constitutive levels, indicating inverse agonism of this compound in this system. The RT-PCR distribution of all known human C-terminal splice variants in human brain regions and periphery showed complex patterns of variant expression, with the novel variant h5-HT4(n) being widely and abundantly expressed.