Illuminating somatostatin analog action at neuroendocrine tumor receptors

Somatostatin analogs for the diagnosis and therapy of neuroendocrine tumors (NETs) have been used in clinical applications for more than two decades. Five somatostatin receptor subtypes have been identified and molecular mechanisms of somatostatin receptor signaling and regulation have been elucidated. These advances increased understanding of the biological role of each somatostatin receptor subtype, their distribution in NETs, as well as agonist-specific regulation of receptor signaling, internalization, and phosphorylation, particularly for the sst2 receptor subtype, which is the primary target of current somatostatin analog therapy for NETs. Various hypotheses exist to explain differences in patient responsiveness to somatostatin analog inhibition of tumor secretion and growth as well as differences in the development of tumor resistance to therapy. In addition, we now have a better understanding of the action of both first generation (octreotide, lanreotide, Octreoscan) and second generation (pasireotide) FDA-approved somatostatin analogs, including the biased agonistic character of some agonists. The increased understanding of somatostatin receptor pharmacology provides new opportunities to design more sophisticated assays to aid the future development of somatostatin analogs with increased efficacy.

P2X7 receptors mediate resistance to toxin-induced cell lysis

In the majority of cells, the integrity of the plasmalemma is recurrently compromised by mechanical or chemical stress. Serum complement or bacterial pore-forming toxins can perforate the plasma membrane provoking uncontrolled Ca(2+) influx, loss of cytoplasmic constituents and cell lysis. Plasmalemmal blebbing has previously been shown to protect cells against bacterial pore-forming toxins. The activation of the P2X7 receptor (P2X7R), an ATP-gated trimeric membrane cation channel, triggers Ca(2+) influx and induces blebbing. We have investigated the role of the P2X7R as a regulator of plasmalemmal protection after toxin-induced membrane perforation caused by bacterial streptolysin O (SLO). Our results show that the expression and activation of the P2X7R furnishes cells with an increased chance of surviving attacks by SLO. This protective effect can be demonstrated not only in human embryonic kidney 293 (HEK) cells transfected with the P2X7R, but also in human mast cells (HMC-1), which express the receptor endogenously. In addition, this effect is abolished by treatment with blebbistatin or A-438079, a selective P2X7R antagonist. Thus blebbing, which is elicited by the ATP-mediated, paracrine activation of the P2X7R, is part of a cellular non-immune defense mechanism. It pre-empts plasmalemmal damage and promotes cellular survival. This mechanism is of considerable importance for cells of the immune system which carry the P2X7R and which are specifically exposed to toxin attacks.

Sheep macrophages express at least two distinct receptors for IgG which have similar affinity for homologous IgG1 and IgG2.

Ovine bone marrow-derived macrophages (BMM) may express several IgG receptor (Fc gamma receptor; FcR) subsets. To study this, model particles (opsonized erythrocytes; EA), which are selectively handled by certain FcR subsets of human macrophages were used in cross-inhibition studies and found to react in a similar manner with FcR subsets of sheep macrophages. In experiments with monoclonal antibodies against subsets of human FcR, human erythrocytes (E) treated with human anti-D-IgG (anti-D-EAhu) and sheep E treated with bovine IgG1 (Bo1-EAs) were handled selectively by human macrophage FcRI and FcRII, respectively. Rabbit-IgG-coated sheep E (Rb-EAs) were recognized by FcRI, FcRII and possibly also by FcRIII of human macrophages. Anti-D-EAhu, Bo1-EAs and Rb-EAs were also ingested by sheep BMM. Competitive inhibition tests, using various homologous and heterologous IgG isotypes as fluid phase inhibitors and the particles used as FcR-specific tools in man (anti-D-EAhu and Bo1-EAs), revealed a heterogeneity of FcR also in sheep BMM. Thus, ingestion of anti-D-EAhu by ovine BMM was inhibited by low concentrations of competitor IgG from rabbit or man in the fluid phase, but not at all by bovine IgG1, whereas ingestion of Bo1-EAs was inhibited by bovine IgG1. This suggested that anti-D-EAhu were recognized by a FcR subset distinct from that recognizing bovine-IgG1. It was concluded that sheep BMM express functional analogs of human macrophage FcRI and FcRII and that Bo1-EAs and anti-D-EAhu are handled by distinct subsets of BMM FcR. All EAhu tested (EAhu treated with anti-D, sheep IgG1 or sheep IgG2) were ingested to a lower degree than EAs. This inefficient phagocytosis could be enhanced by treatment of EAhu with antiglobulin from the rabbit, suggesting that it is caused by a low degree of activity of opsonizing antibodies rather than special properties of the erythrocytes themselves. Several lines of evidence suggested that both FcR subsets of ovine BMM recognize both ovine IgG1 and IgG2. In contrast, bovine IgG1 reacts with one FcR subset and bovine IgG2 interacts inefficiently with all FcR of ovine BMM.

Interactions between Siglec-7/9 receptors and ligands influence NK cell-dependent tumor immunosurveillance

Abstract Alteration of the surface glycosylation pattern on malignant cells potentially affects tumor immunity by directly influencing interactions with glycan-binding proteins (lectins) on the surface of immunomodulatory cells. The sialic acid-binding Ig-like lectins Siglec-7 and -9 are MHC class I-independent inhibitory receptors on human NK cells that recognize sialic acid-containing carbohydrates. Here, we found that the presence of Siglec-9 defined a subset of cytotoxic NK cells with a mature phenotype and enhanced chemotactic potential. Interestingly, this Siglec-9+ NK cell population was reduced in the peripheral blood of cancer patients. Broad analysis of primary tumor samples revealed that ligands of Siglec-7 and -9 were expressed on human cancer cells of different histological types. Expression of Siglec-7 and -9 ligands was associated with susceptibility of NK cell-sensitive tumor cells and, unexpectedly, of presumably NK cell-resistant tumor cells to NK cell-mediated cytotoxicity. Together, these observations have direct implications for NK cell-based therapies and highlight the requirement to consider both MHC class I haplotype and tumor-specific glycosylation.

A novel open channel blocker of GABA-A receptors

GABA-A receptors are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. 19 different subunit isoforms have been identified, with the major receptor type in mammalian adult brain consisting of α1, β2, and γ2 subunits. GABA-A receptors are the target of numerous sedating and anxiolytic drugs such as benzodiazepines. The currently known endogenous ligands are GABA, neurosteroids and the endocannabinoid 2- arachidonoyl glycerol (2-AG). The pharmacological properties of this chloride ion channel strictly depend on receptor subunit composition and arrangement. GABA-A receptors bind and are inhibited by epileptogenic agents such as picrotoxin, and cyclodiene insecticides such as dieldrin. We screened aromatic monovalent anions with five-fold symmetry for inhibition of GABA-A receptors. One of the anions, PCCPinhibited currents elicited by GABA with comparable potency as picrotoxin. This inhibition showed all characteristics of an open channel block. The GABA-A receptor ion channel is lined by residues from the M2 membrane-spanning segment. To identify important residues of the pore involved in the interaction with the blocking molecules PCCP-, a mutation scan was performed in combination with subsequent analysis of the expressed mutant proteins using electrophysiological techniques. In a second project we characterised a light-switchable modulator of GABA-A receptors based on propofol. It was my responsibility to investigate the switching kinetics in patch clamp experiments. After its discovery in 1980, propofol has become the most widely used intravenous general anaesthetic. It is commonly accepted that the anaesthesia induced by this unusually lipophilic drug mostly results from potentiation of GABA induced currents. While GABA-A receptors respond to a variety of ligands, they are normally not sensitive towards light. This light sensitivity could be indirectly achieved by using modulators that can be optically switched between an active and an inactive form. We tested an azobenzene derivative of propofol where an aryldiazene unit is directly coupled to the pharmacophore. This molecule was termed azopropofol (AP2). The effect of AP2 on Cl- currents was investigated with electrophysiological techniques using α1β2γ2 GABA-A receptors expressed in Xenopus oocytes and HEK-cells. In the third project we wanted to investigate the functional role of GABA-A receptors in the liver, and their possible involvement in cell proliferation. GABA-A receptors are also found in a wide range of peripheral tissues, including parts of the peripheral nervous system and non-neural tissues such as smooth muscle, the female reproductive system, liver and several cancer tissues. However their precise function in non neuronal or cancerous cells is still unknown. For this purpose we investigated expression, localization and function of the hepatocytes GABA-A receptors in model cell lines and healthy and cancerous hepatocytes.

The Antimalarial Drug Proguanil Is An Antagonist at 5-HT3 Receptors

Proguanil is an antimalarial prodrug that is metabolized to 4-chlorophenyl-1-biguanide (CPB) and the active metabolite cycloguanil (CG). These compounds are structurally related to meta-chlorophenyl biguanide (mCPBG), a 5-hydroxytryptamine 3 (5-HT3) receptor agonist. Here we examine the effects of proguanil and its metabolites on the electrophysiology and ligand-binding properties of human 5-HT3A receptors expressed in Xenopus oocytes and human embryonic kidney 293 cells, respectively. 5-HT3 receptor responses were reversibly inhibited by proguanil, with an IC50 of 1.81 μM. Competitive antagonism was shown by a lack of voltage-dependence, Schild plot (Kb = 1.70 μM), and radioligand competition (Ki = 2.61 μM) with the 5-HT3 receptor antagonist [3H]granisetron. Kinetic measurements (kon = 4.0 × 104 M−1 s−1; koff = 0.23 s−1) were consistent with a simple bimolecular reaction scheme with a Kb of 4.35 μM. The metabolites CG and CPB similarly inhibited 5-HT3 receptors as assessed by IC50 (1.48 and 4.36 μM, respectively), Schild plot (Kb = 2.97 and 11.4 μM), and radioligand competition (Ki = 4.89 and 0.41 μM). At higher concentrations, CPB was a partial agonist (EC50 = 14.1 μM; I/Imax = 0.013). These results demonstrate that proguanil competitively inhibits 5-HT3 receptors, with an IC50 that exceeds whole-blood concentrations following its oral administration. They may therefore be responsible for the occasional gastrointestinal side effects, nausea, and vomiting reported following its use. Clinical development of related compounds should therefore consider effects at 5-HT3 receptors as an early indication of possible unwanted gastrointestinal side effects.

The antimalarial drugs quinine, chloroquine and mefloquine are antagonists at 5-HT 3 receptors

Background and Purpose: The antimalarial compounds quinine, chloroquine and mefloquine affect the electrophysiological properties of Cys-loop receptors and have structural similarities to 5-HT3 receptor antagonists. They may therefore act at 5-HT3 receptors. Experimental Approach: The effects of quinine, chloroquine and mefloquine on electrophysiological and ligand binding properties of 5-HT3A receptors expressed in HEK 293 cells and Xenopus oocytes were examined. The compounds were also docked into models of the binding site. Key Results: 5-HT3 responses were blocked with IC50 values of 13.4 μM, 11.8 μM and 9.36 μM for quinine, chloroquine and mefloquine. Schild plots indicated quinine and chloroquine behaved competitively with pA2 values of 4.92 (KB=12.0 μM) and 4.97 (KB=16.4 μM). Mefloquine displayed weakly voltage-dependent, non-competitive inhibition consistent with channel block. On and off rates for quinine and chloroquine indicated a simple bimolecular reaction scheme. Quinine, chloroquine and mefloquine displaced [3H]granisetron with Ki values of 15.0, 24.2 and 35.7 μM. Docking of quinine into a homology model of the 5-HT3 receptor binding site located the tertiary ammonium between W183 and Y234, and the quinoline ring towards the membrane, stabilised by a hydrogen bond with E129. For chloroquine, the quinoline ring was positioned between W183 and Y234 and the tertiary ammonium stabilised by interactions with F226. Conclusions and Implications: This study shows that quinine and chloroquine competitively inhibit 5-HT3 receptors, while mefloquine inhibits predominantly non-competitively. Both quinine and chloroquine can be docked into a receptor binding site model, consistent with their structural homology to 5-HT3 receptor antagonists.

Inheritance of porcine receptors for enterotoxigenic Escherichia coli with fimbriae F4ad and their relation to other F4 receptors.

Enteric Escherichia coli infections are a highly relevant cause of disease and death in young pigs. Breeding genetically resistant pigs is an economical and sustainable method of prevention. Resistant pigs are protected against colonization of the intestine through the absence of receptors for the bacterial fimbriae, which mediate adhesion to the intestinal surface. The present work aimed at elucidation of the mode of inheritance of the F4ad receptor which according to former investigations appeared quite confusing. Intestines of 489 pigs of an experimental herd were examined by a microscopic adhesion test modified in such a manner that four small intestinal sites instead of one were tested for adhesion of the fimbrial variant F4ad. Segregation analysis revealed that the mixed inheritance model explained our data best. The heritability of the F4ad phenotype was estimated to be 0.7±0.1. There are no relations to the strong receptors for variants F4ab and F4ac. Targeted matings allowed the discrimination between two F4ad receptors, that is, a fully adhesive receptor (F4adRFA) expressed on all enterocytes and at all small intestinal sites, and a partially adhesive receptor (F4adRPA) variably expressed at different sites and often leading to partial bacterial adhesion. In pigs with both F4ad receptors, the F4adRPA receptor is masked by the F4adRFA. The hypothesis that F4adRFA must be encoded by at least two complementary or epistatic dominant genes is supported by the Hardy-Weinberg equilibrium statistics. The F4adRPA receptor is inherited as a monogenetic dominant trait. A comparable partially adhesive receptor for variant F4ab (F4abRPA) was also observed but the limited data did not allow a prediction of the mode of inheritance. Pigs were therefore classified into one of eight receptor phenotypes: A1 (F4abRFA/F4acR+/F4adRFA); A2 (F4abRFA/F4acR+/F4adRPA); B (F4abRFA/F4acR+/F4adR-); C1 (F4abRPA/F4acR-/F4adRFA); C2 (F4abRPA/F4acR-/F4adRPA); D1 (F4abR-/F4acR-/F4adRFA); D2 (F4abR-/F4acR-/F4adRPA); E (F4abR-/F4acR-/F4adR-).

Fibrinogen association with human monocytes: evidence for constitutive expression of fibrinogen receptors and for involvement of Mac-1 (CD18, CR3) in the binding

Radiolabeled fibrinogen (Fg) specifically binds to mononuclear leukocytes (MNL) and to purified monocytes, but not to nylon-nonadherent lymphocytes. The association is rapid, Ca++-dependent and reversible. MNL containing Fg-binding monocytes had not been exposed to endotoxin (less than 4 pg/mL) during the isolation and the binding test, and Fg binding was not altered by preincubation of MNL with lipopolysaccharide. The binding of Fg was inhibited by anti-Mac-1 antibodies (OKM1). Antibodies to surface-bound Fg were able to induce luminol-dependent chemiluminescence, indicating that Fg binding sites have receptor function. Emission of a signal depended on MNL exposure to Fg, on specific, divalent antibodies, but not on the antibody Fc portion. These data show that human monocytes constitutively express specific Fg receptors and suggest that Mac-1, a member of the integrin superfamily, is involved in Fg recognition.