Messenger RNA levels and binding sites of muscarinic acetylcholine receptors in gastrointestinal muscle layers from healthy dairy cows

Acetylcholine interacts with muscarinic receptors (M) to mediate gastrointestinal (GI) smooth muscle contractions. We have compared mRNA levels and binding sites of M(1)to M(5) in muscle tissues from fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows. The mRNA levels were measured by quantitative RT-PCR. The inhibition of [(3)H]-QNB (1-quinuclidinyl-[phenyl-4-(3)H]-benzilate) binding by M antagonists [atropine (M(1 - 5)), pirenzepine (M(1)), methoctramine (M(2)), 4-DAMP (M(3)), and tropicamide (M(4))] was used to identify receptors at the functional level. Maximal binding (B(max)) was determined through saturation binding with atropine as a competitor. The mRNA levels of M(1), M(2), M(3), and M(5) represented 0.2, 48, 50, and 1.8%, respectively, of the total M population, whereas mRNA of M(4) was undetectable. The mRNA levels of M(2) and of M(3) in the ileum were lower (P < 0.05) than in other GI locations, which were similar among each other. Atropine, pirenzepine, methoctramine, and 4-DAMP inhibited [(3)H]-QNB binding according to an either low- or high-affinity receptor pattern, whereas tropicamide had no effect on [(3)H]-QNB binding. The [(3)H]-QNB binding was dose-dependent and saturable. B(max) in fundus, pylorus, and PLAC was lower (P < 0.05) than in the ELSC, and in the pylorus lower (P < 0.05) than in the ileum. B(max) and mRNA levels were negatively correlated (r = -0.3; P < 0.05). In conclusion, densities of M are different among GI locations, suggesting variable importance of M for digestive functions along the GI tract.

Effects of dexamethasone on mRNA abundance of nuclear receptors and hepatic nuclear receptor target genes in neonatal calves

Hepatic nuclear receptors (NR), particularly constitutive androstane receptor (CAR) and pregnane X receptor (PXR), are involved in the coordinated transcriptional control of genes that encode proteins involved in the metabolism and detoxification of xeno- and endobiotics. A broad spectrum of metabolic processes are mediated by NR acting in concert with ligands such as glucocorticoids. This study examined the role of dexamethasone on hepatic mRNA expression of CAR, PXR and several NR target genes. Twenty-eight male calves were allotted to one of four treatment groups in a 2 x 2 arrangement of treatments: feed source (colostrum or milk-based formula) and glucocorticoid administration (twice daily intramuscular dexamethasone). Liver biopsies were obtained at 5 days of age. Real-time reverse transcription polymerase chain reaction was used to quantify mRNA abundances. No effects of feed source on mRNA abundances were observed. For the NR examined, mRNA abundance of both CAR and PXR in dexamethasone-treated calves was lower (p < 0.05) by 39% and 40%, respectively, than in control calves. Abundance of NR target genes exhibited a mixed response. SULT1A1 mRNA abundance was 39% higher (p < 0.05) in dexamethasone-treated calves compared with control calves. mRNA abundance of CYP2C8 tended also to be higher (+44%; p = 0.053) after dexamethasone treatment. No significant treatment effects (p > 0.10) were observed for mRNA abundances of CYP3A4, CYP2E1, SULT2A1, UGT1A1 or cytochrome P450 reductase (CPR). In conclusion, an enhanced glucocorticoid status, induced by pharmacological amounts of dexamethasone, had differential and in part unexpected effects on NR and NR target systems in 5-day-old calves. Part of the unexpected responses may be due the immaturity of NR and NR receptor target systems.

NPY receptors in human cancer: a review of current knowledge

Many peptide hormone receptors are over-expressed in human cancer, permitting an in vivo targeting of tumors for diagnostic and therapeutic purposes. NPY receptors are novel and promising candidates in this field. Using in vitro receptor autoradiography, Y1 and Y2 receptors have been found to be expressed in breast carcinomas, adrenal gland and related tumors, renal cell carcinomas, and ovarian cancers in both tumor cells and tumor-associated blood vessels. Pathophysiologically, tumoral NPY receptors may be activated by endogenous NPY released from intratumoral nerve fibers or tumor cells themselves, and mediate NPY effects on tumor cell proliferation and tumoral blood supply. Clinically, tumoral NPY receptors may be targeted with NPY analogs coupled with adequate radionuclides or cytotoxic agents for a scintigraphic tumor imaging and/or tumor therapy.

Neurokinin-2 receptor levels correlate with intensity, frequency, and duration of pain in chronic pancreatitis

OBJECTIVE: Generation and maintenance of pain in chronic pancreatitis (CP) have been shown to be partially attributable to neuroimmune interactions, which involve neuropeptides such as substance P (SP). So far, expression of SP receptors NK-2R, NK-3R, the SP-encoding gene preprotachykinin A (PPT-A), and the SP degradation enzyme neutral endopeptidase (NEP) and their relation to pain in CP have not been determined. METHODS: Tissue samples from patients with CP (n = 25) and from healthy donors (n = 20) were analyzed for PPT-A, NK-2R, NK-3R, and NEP expression using quantitative RT-PCR. NEP protein levels were examined by immunoblot analysis and its localization was determined using immunohistochemistry. A scoring system was used to grade the extent of fibrosis on hematoxylin and eosin- and Masson-Trichrome-stained sections. Messenger RNA levels and the extent of pain were analyzed for correlations. RESULTS: In CP tissues, NK-2R and PPT-A expression was increased, whereas NK-3R and NEP mRNA levels were comparable with normal pancreas. Overexpression of NK-2R was related to the intensity, frequency, and duration of pain in CP patients. NK-1R and NEP expression was significantly related to the extent of fibrosis. CONCLUSIONS: Expression of NK-2R and PPT-A is increased in CP and is associated with pain. Failure to up-regulate NEP may contribute to the disruption of the neuropeptides loop balance in CP and thus may exacerbate the severe pain syndrome.

Stable transduction of bovine TLR4 and bovine MD-2 into LPS-nonresponsive cells and soluble CD14 promote the ability to respond to LPS

The interaction of bovine cells with lipopolysaccharide (LPS) was explored using human embryo kidney (HEK) 293 cell line stably transduced with bovine toll-like receptor-4 (TLR4) alone or in combination with bovine MD-2. These lines and mock-transduced HEK293 cells were tested by flow cytometry for LPS-fluorescein isothiocyanate (LPS-FITC) binding, nuclear factor kappa B (NFkappaB) activation, interleukin-8 (IL-8) production and interferon-beta mRNA expression/interferon (IFN) type I production. Whereas bovine TLR4 was sufficient to promote binding of high concentrations of LPS-FITC, both bovine TLR4 and MD-2 were required for activation by LPS, as assessed by NFkappaB activation and IL-8 production. Induction of IFN bioactivity was not observed in doubly transduced HEK293 cells, and no evidence for IFN-beta mRNA induction in response to LPS was obtained, although cells responded by IFN-beta mRNA expression to stimulation by Sendai virus and poly-inosinic acid-poly-cytidylic acid (poly(I:C)). Cells stably transduced with both bovine TLR4 and bovine MD-2 responded to LPS by IL-8 production, in decreasing order, in the presence of fetal bovine serum (FCS), of human serum, and of human serum albumin (HSA). The reduced activity in the presence of HSA could be restored by the addition of soluble CD14 (sCD14) but not of LPS binding protein (LBP). This is in contrast to macrophages which show a superior response to LPS in the presence of HSA when compared with macrophages stimulated by LPS in the presence of FCS. This suggests that macrophages but not HEK293 cells express factors rendering LPS stimulation serum-independent. Stably double-transduced cells reacted, in decreasing order, to LPS from Rhodobacter sphaeroides, to LPS from Escherichia coli, to synthetic lipd-IVa (compound 406), to diphosphoryl-lipid-A (S. minnesota) and to monophosphoryl-lipid-A (S. minnesota). They failed to react to the murine MD-2/TLR4 ligand taxol. This resembles the reactivity of bovine macrophages with regard to sensitivity (ED(50)) and order of potency but is distinct from the reactivity pattern of other species. This formally establishes that in order to react to LPS, cattle cells require serum factors (e.g. sCD14) and cell-expressed factors such as MD-2 and TLR4. The cell lines described are the first of a series expressing defined pattern recognition receptors (PRR) of bovine origin. They will be useful in the study of the interaction of the bovine TLR4-MD-2 complex and Gram-negative bovine pathogens, e.g. the agents causing Gram-negative bovine mastitis.

Impaired protein stability of 11beta-hydroxysteroid dehydrogenase type 2: a novel mechanism of apparent mineralocorticoid excess

Apparent mineralocorticoid excess (AME) is a severe form of hypertension that is caused by impaired activity of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which converts biologically active cortisol into inactive cortisone. Mutations in HSD11B2 result in cortisol-induced activation of mineralocorticoid receptors and cause hypertension with hypokalemia, metabolic alkalosis, and suppressed circulating renin and aldosterone concentrations. This study uncovered the first patient with AME who was described in the literature, identified the genetic defect in HSD11B2, and provided evidence for a novel mechanism of reduced 11beta-HSD2 activity. This study identified a cluster of amino acids (335 to 339) in the C-terminus of 11beta-HSD2 that are essential for protein stability. The cluster includes Tyr(338), which is mutated in the index patient, and Arg(335) and Arg(337), previously reported to be mutated in hypertensive patients. It was found that wild-type 11beta-HSD2 is a relatively stable enzyme with a half-life of 21 h, whereas that of Tyr(338)His and Arg(337)His was 3 and 4 h, respectively. Enzymatic activity of Tyr(338)His was partially retained at 26 degrees C or in the presence of the chemical chaperones glycerol and dexamethasone, indicating thermodynamic instability and misfolding. The results provide evidence that the degradation of both misfolded mutant Tyr(338)His and wild-type 11beta-HSD2 occurs through the proteasome pathway. Therefore, impaired 11beta-HSD2 protein stability rather than reduced gene expression or loss of catalytic activity seems to be responsible for the development of hypertension in some individuals with AME.

11beta-Hydroxysteroid dehydrogenase type 2 in pregnancy and preeclampsia

Cortisol availability is controlled by 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which inactivates cortisol in cortisone, unable to bind to the glucocorticoid receptor. The 11beta-HSD2 enzyme activity limits either intracellular cortisol concentrations or within the uteroplacental compartment the transfer of cortisol into the fetal circulation. Mechanisms, by which 11beta-HSD2 activity is controlled, include transcriptional control, posttranscriptional modifications of 11beta-HSD2 transcript half-life, epigenetic regulation via methylation of genomic DNA and direct inhibition of enzymatic activity. The 11beta-HSD2 expression and activity is reduced in preeclampsia and the enzyme activity correlates with factors associated with increased vasoconstriction, such as an increased angiotensin II receptor subtype 1 expression, and notably fetal growth. Numerous signals such as proinflammatory cytokines known to be present and/or elevated in preeclampsia regulate 11beta-HSD2 activity. Shallow trophoblast invasion with the resulting hypoxemia seems to critically reduce available 11beta-HSD2 activity. A positive feedback exists as activated glucocorticoid receptors do enhance 11beta-HSD2 mRNA transcription and mRNA stability. No data are currently available on pregnancy and either epigenetic or direct effects on the activity of the translated enzyme.

Increased invasive potential and up-regulation of MMP-2 in MDA-MB-231 breast cancer cells expressing the beta3 integrin subunit

Integrins are a family of transmembrane adhesion receptors that might transduce signals from the extracellular matrix into the inside of cells after ligand binding. In order to investigate whether beta3 integrins expressed in tumor cells might mediate such outside-in signaling, human MDA-MB-231 breast cancer cells that were stably transfected with either beta3 integrin or mock-transfected were investigated in a matrigel degradation assay and a grafting experiment was performed on the developing chicken chorioallantoic membrane (CAM). After cultivation on matrigel for time periods between one and five days, more matrigel was digested in the wells in which beta3 integrin expressing cells were incubated than in wells of mock-transfected cells. Furthermore, extracts of beta3 integrin expressing cells contained higher levels of MMP-2 protein as determined by immunoblotting and more MMP-2 associated gelatinase activity as detected by zymography than extracts of mock-transfected cells. Matrigel degradation and gelatinase activity as well as MMP-2 expression were elevated when beta3 integrin expressing cells were incubated in the presence of the RGD peptide (mimicking an integrin ligand). After grafting on 10 day-old embryonic chicken CAM for three to five days, beta3 integrin expressing cells assembled in spheroids showed higher rates of spreading on the CAM surface and CAM invasion as well as a significant MMP-2 up-regulation compared to mock-transfected cells. The results from the in vivo and in vitro experiments allow the conclusion that the presence of beta3 integrin in MDA-MB-231 breast cancer cells induced an increased MMP-2 expression and activity that might contribute to the enhanced invasive potential observed.

Influence of inhibitory killer immunoglobulin-like receptors and their HLA-C ligands on resolving hepatitis C virus infection

An estimated 2%-3% of the world's population is chronically infected with hepatitis C virus (HCV) and this is a major cause of liver disease worldwide. Following acute infection, outcome is variable with acute HCV successfully resolved in some individuals (20%-30%), but in the majority of cases the virus is able to persist. Co-infection with human immunodeficiency virus has been associated with a negative impact on the course of HCV infection. The host's immune response is an important correlate of HCV infection outcome and disease progression. Natural killer (NK) cells provide a major component of the antiviral immune response by recognising and killing virally infected cells. NK cells modulate their activity through a combination of inhibitory and activatory receptors such as the killer immunoglobulin-like receptors (KIRs) that bind to human leukocyte antigen (HLA) Class I molecules. In this workshop component, we addressed the influence of KIR genotypes and their HLA ligands on resolving HCV infection and we discuss the implications of the results of the study of Lopez-Vazquez et al. on KIR and HCV disease progression.

Nuclear localization of epidermal growth factor and epidermal growth factor receptors in human thyroid tissues

Epidermal growth factor (EGF) has widespread growth effects, and in some tissues proliferation is associated with the nuclear localization of EGF and epidermal growth factor receptor (EGFR). In the thyroid, EGF promotes growth but differs from thyrotropin (TSH) in inhibiting rather than stimulating functional parameters. We have therefore studied the occurrence and cellular distribution of EGF and EGFR in normal thyroid, in Graves' disease, where growth is mediated through the thyrotropin receptor (TSHR), and in a variety of human thyroid tumors. In the normal gland the staining was variable, but largely cytoplasmic, for both EGF and EGFR. In Graves' disease there was strong cytoplasmic staining for both EGF and EGFR, with frequent positive nuclei. Nuclear positivity for EGF and particularly for EGFR was also a feature of both follicular adenomas and follicular carcinomas. Interestingly, nuclear staining was almost absent in papillary carcinomas. These findings document for the first time the presence of nuclear EGF and EGFR in thyroid. Their predominant occurrence in tissues with increased growth (Graves' disease, follicular adenoma, and carcinoma) may indicate that nuclear EGF and EGFR play a role in growth regulation in these conditions. The absence of nuclear EGF and EGFR in papillary carcinomas would suggest that the role played by EGF in growth control differs between papillary carcinoma and follicular adenomas/carcinomas of the thyroid.

Neutrophil apoptosis mediated by nicotinic acid receptors (GPR109A)

G protein-coupled receptor (GPR)109A (HM74A) is a G(i) protein-coupled receptor, which is activated by nicotinic acid (NA), a lipid-lowering drug. Here, we demonstrate that mature human neutrophils, but not eosinophils, express functional GPR109A receptors. The induction of the GPR109A gene appears to occur late in the terminal differentiation process of neutrophils, since a mixed population of immature bone marrow neutrophils did not demonstrate evidence for its expression. NA accelerated apoptosis in cultured neutrophils in a concentration-dependent manner, as assessed by phosphatidylserine redistribution, caspase-3 activation, and DNA fragmentation assays. The pro-apoptotic effect of NA was abolished by pertussis toxin, which was used to block G(i) proteins, suggesting a receptor-mediated mechanism. Activation of GPR109A by NA resulted in decreased levels of cyclic adenosine monophosphate (cAMP), most likely due to G(i)-mediated inhibition of adenylyl cyclase activity. NA-induced apoptosis was reversed by the addition of cell-permeable cAMP, pointing to the possibility that reduced cAMP levels promote apoptosis in neutrophils. Distal mechanism involved in this process may include the post-translational modification of members of the Bcl-2 family, such as dephosphorylation of pro-apoptotic Bad and antiapoptotic Mcl-1 proteins. Taken together, following maturation in the bone marrow, neutrophils express functional GPR109A receptors, which might be involved in the regulation of neutrophil numbers. Moreover, this study identified a new cellular target of NA and future drugs activating GPR109A receptors, the mature neutrophil.

Soluble tumour necrosis factor receptors sTNFR1 and sTNFR2 are produced at sites of inflammation and are markers of arthritis activity in Behçet's disease

OBJECTIVE: We analysed the production of soluble tumour necrosis factor receptors sTNFR1 and sTNFR2 at sites of inflammation and measured their plasma concentrations to evaluate them as biological markers of disease activity. METHODS: Plasma samples of 35 patients with Behçet's disease (BD) were collected prospectively at monthly intervals and grouped for inactive disease, active BD without arthritis, and active BD with arthritis. sTNFR1 and sTNFR2 concentrations were measured using immunoassays and compared with other biological disease activity parameters. Plasma sTNFR levels were compared to synovial fluid (SF) levels in seven patients. Sixteen tissue samples of mucocutaneous lesions were stained for TNFR2 expression by immunohistochemistry. RESULTS: sTNFR1 and sTNFR2 were found at increased plasma concentrations in active BD, with the highest concentration in active BD with arthritis (p<0.001). Concentrations of both sTNFRs were at least three times higher in SF of arthritic joints than in the corresponding plasma samples (p = 0.025). A change of more than 1 ng/mL of sTNFR2 plasma concentrations correlated with a concordant change in arthritic activity (96% confidence interval). Sensitivity to change was superior to that of sTNFR1, and other biological disease activity parameters such as erythrocyte sedimentation rate (ESR), immunoglobulin (Ig)G, IgA, and interleukin (IL)-10 plasma concentrations. A strong staining for TNFR2 was found in mucocutaneous lesions, where mast cells were identified as the major source for this receptor. CONCLUSIONS: This longitudinal study demonstrates that sTNFR2 plasma concentrations are closely linked with active BD, and especially with arthritis. Taken together with the expression of TNFR molecules in mast cells of mucocutaneous lesions, our results indicate a fundamental role for the TNF/TNFR pathway in BD.

Toll-like receptor 2 is highly expressed in lesions of acne inversa and colocalizes with C-type lectin receptor

BACKGROUND: Acne inversa (hidradenitis suppurativa) is a chronic inflammatory and cicatricial disorder that affects skin areas rich in apocrine glands and terminal hairs, such as perineum and axillae. The exact pathogenesis of the disease is not well understood and the mechanisms by which bacterial superinfection contributes to the disease progression are not clear. Toll-like receptors (TLRs) expressed by inflammatory cells play a crucial role in the innate immune response to bacteria. OBJECTIVES: We sought to investigate the role of TLR2 in the pathogenesis of acne inversa. METHODS: We investigated the expression of TLR2 using real-time polymerase chain reaction analysis and immunohistochemical stainings of tissue samples from patients with acne inversa. Furthermore, we phenotypically characterized the infiltrating cells and their expression of TLR2. RESULTS: Compared with normal skin, a highly increased in situ expression of TLR2 in acne inversa skin lesions was found at both the mRNA and the protein level. The most abundant cells in the dermal infiltrate of acne inversa were CD68+ macrophages, CD209+ dendritic cells (DCs) and CD3+ T cells. CD19+ B cells and CD56+ natural killer cells were found only in small numbers. Double staining with fluorescence-labelled antibodies showed that TLR2 was expressed by infiltrating macrophages (CD68+) and DCs (CD209+). Flow cytometric analysis of isolated infiltrating cells further confirmed surface expression of TLR2 by macrophages and DCs. CONCLUSIONS: These data indicate that the enhanced expression of TLR2 by infiltrating macrophages and DCs may contribute to the pathogenesis of inflammatory lesions of acne inversa.

Alkylamides from Echinacea are a new class of cannabinomimetics. Cannabinoid type 2 receptor-dependent and -independent immunomodulatory effects

Alkylamides (alkamides) from Echinacea modulate tumor necrosis factor alpha mRNA expression in human monocytes/macrophages via the cannabinoid type 2 (CB2) receptor (Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563-569). Here we show that the alkylamides dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and dodeca-2E,4E-dienoic acid isobutylamide (A2) bind to the CB2 receptor more strongly than the endogenous cannabinoids. The Ki values of A1 and A2 (CB2 approximately 60 nM; CB1 >1500 nM) were determined by displacement of the synthetic high affinity cannabinoid ligand [3H]CP-55,940. Molecular modeling suggests that alkylamides bind in the solvent-accessible cavity in CB2, directed by H-bonding and pi-pi interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB2 and CB1. A1 and A2 elevated total intracellular Ca2+ in CB2-positive but not in CB2-negative promyelocytic HL60 cells, an effect that was inhibited by the CB2 antagonist SR144528. At 50 nM, A1, A2, and the endogenous cannabinoid anandamide (CB2 Ki >200 nM) up-regulated constitutive interleukin (IL)-6 expression in human whole blood in a seemingly CB2-dependent manner. A1, A2, anandamide, the CB2 antagonist SR144528 (Ki <10 nM), and also the non-CB2-binding alkylamide undeca-2E-ene,8,10-diynoic acid isobutylamide all significantly inhibited lipopolysaccharide-induced tumor necrosis factor alpha, IL-1beta, and IL-12p70 expression (5-500 nM) in a CB2-independent manner. Alkylamides and anandamide also showed weak differential effects on anti-CD3-versus anti-CD28-stimulated cytokine expression in human whole blood. Overall, alkylamides, anandamide, and SR144528 potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects are not exclusively related to CB2 binding.

High expression of gastrin-releasing peptide receptors in the vascular bed of urinary tract cancers: promising candidates for vascular targeting applications

Tumoral gastrin-releasing peptide (GRP) receptors are potential targets for diagnosis and therapy using radiolabeled or cytotoxic GRP analogs. GRP-receptor overexpression has been detected in endocrine-related cancer cells and, more recently, also in the vascular bed of selected tumors. More information on vascular GRP-receptors in cancer is required to asses their potential for vascular targeting applications. Therefore, frequent human cancers (n = 368) were analyzed using in vitro GRP-receptor autoradiography on tissue sections with the (125)I-[Tyr(4)]-bombesin radioligand and/or the universal radioligand (125)I-[d-Tyr(6), beta-Ala(11), Phe(13), Nle(14)]-bombesin(6-14). GRP-receptor expressing vessels were evaluated in each tumor group for prevalence, quantity (vascular score), and GRP-receptor density. Prevalence of vascular GRP-receptors was variable, ranging from 12% (prostate cancer) to 92% (urinary tract cancer). Different tumor types within a given site had divergent prevalence of vascular GRP-receptors (e.g. lung: small cell cancer: 0%; adenocarcinoma: 59%; squamous carcinoma: 83%). Also the vascular score varied widely, with the highest score in urinary tract cancer (1.69), moderate scores in lung (0.91), colon (0.88), kidney (0.84), and biliary tract (0.69) cancers and low scores in breast (0.39) and prostate (0.14) cancers. Vascular GRP-receptors were expressed in the muscular vessel wall in moderate to high densities. Normal non-neoplastic control tissues from these organs lacked vascular GRP-receptors. In conclusion, tumoral vessels in all evaluated sites express GRP-receptors, suggesting a major biological function of GRP-receptors in neovasculature. Vascular GRP-receptor expression varies between the tumor types indicating tumor-specific mechanisms in their regulation. Urinary tract cancers express vascular GRP-receptors so abundantly, that they are promising candidates for vascular targeting applications.