Binding sites of muscarinic and adrenergic receptors in gastrointestinal tissues of dairy cows suffering from left displacement of the abomasum

Muscarinic acetylcholine (M) and adrenergic (AR) receptors mediate gastrointestinal motility. Using radioligand binding assays and real-time polymerase chain reaction, the densities of binding sites and mRNA levels of M(2), M(3), alpha(2AD)- and beta(2)-AR were compared in muscle tissues from the abomasal fundus, pylorus, duodenum, caecum, and external loop of the spiral colon of eight cows with left displacement of abomasum (LDA), and of eight healthy cows. Specific binding of the [(3)H]-ligands to each of the four receptors was competitive and saturable. Binding sites of M(2) (all intestinal sites), M(3) (duodenum and caecum), and of alpha(2AD)-AR (abomasal fundus) were lower (P<0.05) in cows with LDA than in healthy cows. The coefficients of correlation between binding sites and mRNA transcripts of receptors were dissimilar in cows with LDA and healthy cows. The decrease in densities of M (intestine) and of alpha(2AD)-AR (abomasum) receptors suggests their implication in the impairment of motility associated with or leading to LDA.

Toll-like receptors in ischaemia and its potential role in the pathophysiology of muscle damage in critical limb ischaemia

Toll-like receptors (TLRs) are key receptors of the innate immune system which are expressed on immune and nonimmune cells. They are activated by both pathogen-associated molecular patterns and endogenous ligands. Activation of TLRs culminates in the release of proinflammatory cytokines, chemokines, and apoptosis. Ischaemia and ischaemia/reperfusion (I/R) injury are associated with significant inflammation and tissue damage. There is emerging evidence to suggest that TLRs are involved in mediating ischaemia-induced damage in several organs. Critical limb ischaemia (CLI) is the most severe form of peripheral arterial disease (PAD) and is associated with skeletal muscle damage and tissue loss; however its pathophysiology is poorly understood. This paper will underline the evidence implicating TLRs in the pathophysiology of cerebral, renal, hepatic, myocardial, and skeletal muscle ischaemia and I/R injury and discuss preliminary data that alludes to the potential role of TLRs in the pathophysiology of skeletal muscle damage in CLI.

Structure, function, and modulation of GABA(A) receptors

The GABA(A) receptors are the major inhibitory neurotransmitter receptors in mammalian brain. Each isoform consists of five homologous or identical subunits surrounding a central chloride ion-selective channel gated by GABA. How many isoforms of the receptor exist is far from clear. GABA(A) receptors located in the postsynaptic membrane mediate neuronal inhibition that occurs in the millisecond time range; those located in the extrasynaptic membrane respond to ambient GABA and confer long-term inhibition. GABA(A) receptors are responsive to a wide variety of drugs, e.g. benzodiazepines, which are often used for their sedative/hypnotic and anxiolytic effects.

The cannabinoid CB1 receptor antagonists rimonabant (SR141716) and AM251 directly potentiate GABA(A) receptors

Rimonabant (SR141716) and the structurally related AM251 are widely used in pharmacological experiments as selective cannabinoid receptor CB(1) antagonists / inverse agonists. Concentrations of 0.5-10 µM are usually applied in in vitro experiments. We intended to show that these drugs did not act at GABA(A) receptors but found a significant positive allosteric modulation instead.

Identification and quantification of a new family of peptide endocannabinoids (Pepcans) showing negative allosteric modulation at CB1 receptors

The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB(1)). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB(1) receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [(3)H]CP55,940 and [(3)H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [(3)H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB(1) receptors. Competition binding studies revealed K(i) values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [(35)S]GTPγS binding, and CB(1) receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB(1) receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling.

High salt intake down-regulates colonic mineralocorticoid receptors, epithelial sodium channels and 11β-hydroxysteroid dehydrogenase type 2

Besides the kidneys, the gastrointestinal tract is the principal organ responsible for sodium homeostasis. For sodium transport across the cell membranes the epithelial sodium channel (ENaC) is of pivotal relevance. The ENaC is mainly regulated by mineralocorticoid receptor mediated actions. The MR activation by endogenous 11β-hydroxy-glucocorticoids is modulated by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). Here we present evidence for intestinal segment specific 11β-HSD2 expression and hypothesize that a high salt intake and/or uninephrectomy (UNX) affects colonic 11β-HSD2, MR and ENaC expression. The 11β-HSD2 activity was measured by means of 3H-corticosterone conversion into 3H-11-dehydrocorticosterone in Sprague Dawley rats on a normal and high salt diet. The activity increased steadily from the ileum to the distal colon by a factor of about 3, an observation in line with the relevance of the distal colon for sodium handling. High salt intake diminished mRNA and protein of 11β-HSD2 by about 50% (p<0.001) and reduced the expression of the MR (p<0.01). The functionally relevant ENaC-β and ENaC-γ expression, a measure of mineralocorticoid action, diminished by more than 50% by high salt intake (p<0.001). The observed changes were present in rats with and without UNX. Thus, colonic epithelial cells appear to contribute to the protective armamentarium of the mammalian body against salt overload, a mechanism not modulated by UNX.

Impact of estrogen replacement throughout childhood on growth, pituitary-gonadal axis and bone in a 46,XX patient with CYP19A1 deficiency

The adequate replacement dose of estrogens during infancy and childhood is still not known in girls. Aromatase deficiency offers an excellent model to study how much estrogens are needed during infancy, childhood and adulthood.

Transfection of drug-specific T-cell receptors into hybridoma cells: tools to monitor drug interaction with T-cell receptors and evaluate cross-reactivity to related compounds

In the context of drug hypersensitivity, our group has recently proposed a new model based on the structural features of drugs (pharmacological interaction with immune receptors; p-i concept) to explain their recognition by T cells. According to this concept, even chemically inert drugs can stimulate T cells because certain drugs interact in a direct way with T-cell receptors (TCR) and possibly major histocompatibility complex molecules without the need for metabolism and covalent binding to a carrier. In this study, we investigated whether mouse T-cell hybridomas transfected with drug-specific human TCR can be used as an alternative to drug-specific T-cell clones (TCC). Indeed, they behaved like TCC and, in accordance with the p-i concept, the TCR recognize their specific drugs in a direct, processing-independent, and dose-dependent way. The presence of antigen-presenting cells was a prerequisite for interleukin-2 production by the TCR-transfected cells. The analysis of cross-reactivity confirmed the fine specificity of the TCR and also showed that TCR transfectants might provide a tool to evaluate the potential of new drugs to cause hypersensitivity due to cross-reactivity. Recombining the alpha- and beta-chains of sulfanilamide- and quinolone-specific TCR abrogated drug reactivity, suggesting that both original alpha- and beta-chains were involved in drug binding. The TCR-transfected hybridoma system showed that the recognition of two important classes of drugs (sulfanilamides and quinolones) by TCR occurred according to the p-i concept and provides an interesting tool to study drug-TCR interactions and their biological consequences and to evaluate the cross-reactivity potential of new drugs of the same class.

Structure and binding kinetics of three different human CD1d-alpha-galactosylceramide-specific T cell receptors

Invariant human TCR Valpha24-Jalpha18+/Vbeta11+ NKT cells (iNKT) are restricted by CD1d-alpha-glycosylceramides. We analyzed crystal structures and binding characteristics for an iNKT TCR plus two CD1d-alpha-GalCer-specific Vbeta11+ TCRs that use different TCR Valpha chains. The results were similar to those previously reported for MHC-peptide-specific TCRs, illustrating the versatility of the TCR platform. Docking TCR and CD1d-alpha-GalCer structures provided plausible insights into their interaction. The model supports a diagonal orientation of TCR on CD1d and suggests that complementarity determining region (CDR)3alpha, CDR3beta, and CDR1beta interact with ligands presented by CD1d, whereas CDR2beta binds to the CD1d alpha1 helix. This docking provides an explanation for the dominant usage of Vbeta11 and Vbeta8.2 chains by human and mouse iNKT cells, respectively, for recognition of CD1d-alpha-GalCer.

Cardiac glycosides initiate Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cells by up-regulation of death receptors 4 and 5

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the TNF family known to transduce their death signals via cell membrane receptors. Because it has been shown that Apo2L/TRAIL induces apoptosis in tumor cells without or little toxicity to normal cells, this cytokine became of special interest for cancer research. Unfortunately, cancer cells are often resistant to Apo2L/TRAIL-induced apoptosis; however, this can be at least partially negotiated by parallel treatment with other substances, such as chemotherapeutic agents. Here, we report that cardiac glycosides, which have been used for the treatment of cardiac failure for many years, sensitize lung cancer cells but not normal human peripheral blood mononuclear cells to Apo2L/TRAIL-induced apoptosis. Sensitization to Apo2L/TRAIL mediated by cardiac glycosides was accompanied by up-regulation of death receptors 4 (DR4) and 5 (DR5) on both RNA and protein levels. The use of small interfering RNA revealed that up-regulation of death receptors is essential for the demonstrated augmentation of apoptosis. Blocking of up-regulation of DR4 and DR5 alone significantly reduced cell death after combined treatment with cardiac glycosides and Apo2L/TRAIL. Combined silencing of DR4 and DR5 abrogated the ability of cardiac glycosides and Apo2L/TRAIL to induce apoptosis in an additive manner. To our knowledge, this is the first demonstration that glycosides up-regulate DR4 and DR5, thereby reverting the resistance of lung cancer cells to Apo2/TRAIL-induced apoptosis. Our data suggest that the combination of Apo2L/TRAIL and cardiac glycosides may be a new interesting anticancer treatment strategy.

Ontogeny of mRNA abundance of nuclear receptors and nuclear receptor target genes in young cattle

After birth the development of appropriate detoxification mechanisms is important. Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-alpha (PPARalpha), retinoid receptors (RAR, RXR), and NR target genes are involved in the detoxification of exogenous and endogenous substances. We quantified abundances of hepatic mRNA of NR and several NR target genes (cytochromes, CYP; cytochrome P450 reductase, CPR; UDP-glucuronosyl transferase, UDP) in calves at different ages. Gene expression was quantified by real-time RT-PCR. Abundance of mRNA of CAR and PXR increased from low levels at birth in pre-term calves (P0) and full-term calves (F0) to higher levels in 5-day-old calves (F5) and in 159-day-old veal calves (F159), whereas mRNA levels of PPARalpha did not exhibit significant ontogenetic changes. RARbeta mRNA levels were higher in F5 and F159 than in F0, whereas no age differences were observed for RARalpha levels. Levels of RXRalpha and RXRbeta mRNA were lower in F5 than in P0 and F0. Abundance of CYP2C8 and CYP3A4 increased from low levels in P0 and F0 to higher levels in F5 and to highest levels in F159. Abundance of CPR was transiently decreased in F0 and F5 calves. Levels of UGT1A1 mRNA increased from low levels in P0 and F0 to maximal level in F5 and F159. In conclusion, mRNA levels of NR and NR target genes exhibited ontogenetic changes that are likely of importance for handling of xeno- and endobiotics with increasing age.

Secretin receptors in the human liver: expression in biliary tract and cholangiocarcinoma, but not in hepatocytes or hepatocellular carcinoma

BACKGROUND/AIMS: Gut hormone receptors are over-expressed in human cancer and allow receptor-targeted tumor imaging and therapy. A novel promising receptor for these purposes is the secretin receptor. The secretin receptor expression was investigated in the human liver because the liver is a physiological secretin target and because novel diagnostic and treatment modalities are needed for liver cancer. METHODS: Nineteen normal livers, 10 cirrhotic livers, 35 cholangiocarcinomas, and 45 hepatocellular carcinomas were investigated for secretin receptor expression by in vitro receptor autoradiography using (125)I-[Tyr(10)] rat secretin and, in selected cases, for secretin receptor mRNA by RT-PCR. RESULTS: Secretin receptors were present in normal bile ducts and ductules, but not in hepatocytes. A significant receptor up-regulation was observed in ductular reaction in liver cirrhosis. Twenty-two (63%) cholangiocarcinomas were positive for secretin receptors, while hepatocellular carcinomas were negative. RT-PCR revealed wild-type receptor mRNA in the non-neoplastic liver, wild-type and spliced variant receptor mRNAs in cholangiocarcinomas found receptor positive in autoradiography experiments, and no receptor transcripts in autoradiographically negative cholangiocarcinomas. CONCLUSIONS: The expression of secretin receptors in the biliary tract is the molecular basis of the secretin-induced bicarbonate-rich choleresis in man. The high receptor expression in cholangiocarcinomas may be used for in vivo secretin receptor-targeting of these tumors and for the differential diagnosis with hepatocellular carcinoma.

CRF receptors in the rodent and human cardiovascular systems: Species differences

CRF has powerful receptor-mediated cardiovascular actions. To evaluate the precise distribution of CRF receptors, in vitro CRF receptor autoradiography with (125)I-[Tyr(0), Glu(1), Nle(17)]-sauvagine or [(125)I]-antisauvagine-30 was performed in the rodent and human cardiovascular system. An extremely high density of CRF(2) receptors was detected with both tracers in vessels of rodent lung, intestine, pancreas, mesenterium, kidney, urinary bladder, testis, heart, brain, and in heart muscle. In humans, CRF(2) receptors were detected with (125)I- antisauvagine-30 at low levels in vessels of kidneys, intestine, urinary bladder, testis, heart and in heart muscle, while only heart vessels were detected with (125)I-[Tyr(0), Glu(1), Nle(17)]-sauvagine. This is the first extensive morphological study reporting the extremely wide distribution of CRF(2) receptors in the rodent cardiovascular system and a more limited expression in man, suggesting a species-selective CRF receptor expression.

Selective in vitro targeting of GRP and NMB receptors in human tumours with the new bombesin tracer 177Lu-AMBA

PURPOSE: To investigate the in vitro binding properties of a novel radiolabelled bombesin analogue, (177)Lu-AMBA, in human neoplastic and non-neoplastic tissues selected for their expression of the bombesin receptor subtypes GRP-R, NMB-R and BRS-3. METHODS: In vitro receptor autoradiography was performed in cancers expressing the various bombesin receptor subtypes. The novel radioligand (177)Lu-AMBA was used and compared with established bombesin radioligands such as (125)I-Tyr(4)-bombesin and (125)I-[D: -Tyr(6),beta-Ala(11),Phe(13),Nle(14)]-bombesin(6-14). In vitro incidence of detection of each of the three bombesin receptor subtypes was evaluated in each tumour. RESULTS: (177)Lu-AMBA identified all GRP-R-expressing tumours, such as prostatic, mammary and renal cell carcinomas as well as gastrointestinal stromal tumours. (177)Lu-AMBA also identified all NMB-expressing tumours, but did not detect BRS-3-expressing tumours or BRS-3-expressing pancreatic islets. GRP-R-expressing peritumoural vessels were heavily labelled with (177)Lu-AMBA. In contrast to the strongly GRP-R-positive mouse pancreas, the human pancreas was not labelled with (177)Lu-AMBA unless chronic pancreatitis was diagnosed. In general, the sensitivity was slightly better with (177)Lu-AMBA than with the conventional bombesin radioligands. CONCLUSION: The present in vitro study suggests that (177)Lu-AMBA may be a very useful in vivo targeting agent for GRP-R-expressing tumours, NMB-R-expressing tumours and GRP-R-expressing neoangiogenic vessels.