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A N Eberle

Publications and source records attributed to A N Eberle.

At least 19 recordsLinked to original sources
PubMed2001

Modulation of Na/K-ATPase activity by isoproterenol and propranolol in human non-pigmented ciliary epithelial cells.

PURPOSE: The present study investigates whether beta-adrenoreceptor agents such as isoproterenol and propranolol can regulate Na(+)-K(+)-ATPase activity in cultured human non-pigmented ciliary epithelial cells. METHODS: Human non-pigmented ciliary epithelial cells (ODM2) were grown to confluence. The active ion transport mediated by the Na(+)-K(+)-ATPase was evaluated by measuring ouabain-sensitive rubidium (Rb+) uptake. In a first set of experiments, cells were exposed to the beta-adrenoreceptor agonist isoproterenol (0.01-10 microM). In a second set of experiments, cells were exposed to isoproterenol (1 microM) in the presence of different concentrations of the beta-adrenoreceptor antagonist propranolol (0.01, 0.1, 1 microM). RESULTS: In a concentration-dependent manner, isoproterenol induced an increase in Na(+)-K(+)-ATPase activity. The maximal Na(+)-K(+)-ATPase activity was observed at a concentration of 1 microM of isoproterenol (283 +/- 58%, P < 0.001). The increase in Na(+)-K(+)-ATPase activity evoked by isoproterenol (1 microM) was inhibited. In a concentration dependent manner, by propranolol (maximum: 659 +/- 39 vs. 141 +/- 42 pM/mg protein/min, P < 0.01). CONCLUSION: The beta-adrenoreceptor agents isoproterenol and propranolol are apparently able to modulate Na(+)-K(+)-ATPase activity in cultured human non-pigmented ciliary epithelial cells.

Adrenergic beta-Agonists↗
PubMed2001

Antagonist and agonist activities of the mouse agouti protein fragment (91-131) at the melanocortin-1 receptor.

Antagonist and agonist activities of chemically synthetized mouse agouti protein fragment (91-131) (AP91-131) at the melanocortin type-1 receptor (MC1-R) were assessed using B 16-F1 mouse melanoma cells in vitro and the following assay systems: (i) receptor binding, (ii) adenylate cyclase, (iii) tyrosinase, (iv) melanin production, and (v) cell proliferation. In competition binding studies AP91-131 was about 3-fold less potent than the natural agonist alpha-melanocyte-stimulating hormone (alpha-MSH) in displacing the radioligand [125I]-[Nle4, D-Phe7]-alpha-MSH (Ki 6.5 +/- 0.8 nmol/l). Alpha-MSH-induced tyrosinase activation and melanin production were completely inhibited by a 100-fold higher concentration of AP9 l -131; the IC50 values for AP91-131 in thetwo assay systems were 91 +/- 22 nM and 95 +/- 15 nM respectively. Basal melanin production and adenylate cyclase activity in the absence of agonist were decreased by AP91-131 with IC50 values of 9.6+/-1.8 nM and 5.0+/-2.4 nM, respectively. This indicates inverse agonist activity of AP91-131 similar to that of native AP. The presence of 10 nM melanin-concentrating hormone (MCH) slightly potentiated the inhibitory activity of AP91-131 in the adenylate cyclase and melanin assays. On the other hand, AP91-131 inhibited cell growth similar to alpha-MSH (IC50 11.0 +/- 2.1 nM; maximal inhibition 1.8-fold higher than that of alpha-MSH). Furthermore, MC1-R was down-regulated by AP91-131 with about the same potency and time-course as with alpha-MSH. These results demonstrate that AP91-131 displays both agonist and antagonist activities at the MC1-R and hence that it is the cysteine-rich region of agouti protein which inhibits and mimics the different alpha-MSH functions, most likely by simultaneous modulation of different intracellular signalling pathways.

Adenylyl Cyclases↗
PubMed2001

Interaction of melanin-concentrating hormone (MCH), neuropeptide E-I (NEI), neuropeptide G-E (NGE), and alpha-MSH with melanocortin and MCH receptors on mouse B16 melanoma cells.

Melanin-concentrating hormone (MCH) and alpha-melanocyte-stimulating hormone (alpha-MSH) are known to exhibit mostly functionally antagonistic, but in some cases agonistic activities, e.g., in pigment cells and in the brain. Neuropeptide E-I (NEI) displays functional MCH-antagonist and MSH-agonist activity in different behavioral paradigms; the role of neuropeptide G-E (NGE) is not known. This study addressed the question of possible molecular interactions between alpha-MSH, MCH and the MCH-precursor-derived peptides NEI and NGE at the level of the pigment cell MCH receptor subtype (MCH-Rpc) and the different melanocortin (MC) receptors. Radioreceptor assays using [125I]MCH, [125l]alpha-MSH and [125I]NEI as radioligands and bioassays were performed with MCI-R-positive and MC1-R-negative mouse B16 melanoma cells and with COS cells expressing the different MC receptors. The IC50s of alpha-MSH and NEI or NGE for [125I]MCH displacement from mouse MCH-Rpc were 80-fold and, respectively, >300-fold higher than that of MCH, and the IC50s for MCH and NEI or NGE for [125I]alpha-MSH displacement from mouse MC1-R were 50,000-fold and >200,000-fold higher than that of alpha-MSH. No high-affinity binding sites for NEI were detected on B16 melanoma cells and there was no significant displacement of [1251]alpha-MSH by MCH, NEI or NGE with MC3-R, MC4-R and MC5-R expressed in COS cells. At concentrations of 100 nM to 10 microM, however, MCH, NEI and NGE induced cAMP formation and melanin synthesis which could be blocked by agouti protein or inhibitors of adenylate cyclase or protein kinase A. This shows that mammalian MCH-precursor-derived peptides may mimic MSH signalling via MC1-R activation at relatively high, but physiologically still relevant concentrations, as e.g. found in autocrine/paracrine signalling mechanisms.

Animals↗
PubMed2001

Expression of the melanin-concentrating hormone receptor in porcine and human ciliary epithelial cells.

PURPOSE: To evaluate whether the receptors for melanin-concentrating hormone (MCH) and its functional antagonist alpha-melanocyte-stimulating hormone (alpha-MSH) are expressed in the ciliary epithelium. Furthermore, to examine whether MCH, a neuropeptide involved in fluid and electrolyte homeostasis, may influence ion flux mediated by Na,K (adenosine triphosphatase)-ATPase in a ciliary epithelial cell line. METHODS: Expression of MCH receptors (MCH-R) and alpha-MSH receptors (MSH-R) on primary porcine ciliary pigmented epithelial (PE) cells and on a human nonpigmented ciliary epithelial (NPE) cell line, ODM-2 was investigated by radioligand binding studies and reverse transcription-polymerase chain reaction (RT-PCR). The MCH-R was further characterized by photocrosslinking. Influence of MCH on Na, K-ATPase activity was evaluated by an Rb(+) transport assay. RESULTS: MCH-R expression was observed at both the mRNA and protein levels in PE and NPE cells. In contrast, MSH-Rs were not detectable. At the mRNA level, expression of slc-1 was shown and with crosslinking, a 44-kDa protein was labeled. MCH showed no effect on Na,K-ATPase activity of NPE cells. CONCLUSIONS: The presence of MCH-R in ciliary epithelial cells of both human and porcine origin but the absence of MSH-Rs indicates that in these cells, MCH and alpha-MSH do not form a functionally antagonistic hormonal pair as they do in several other systems. Although effects of MCH on intestinal water and ion transport have been documented, a direct control of Na,K-ATPase activity was not detected in human NPE cells in vitro.

Animals↗
PubMed2000

beta-endorphin stimulates cytokeratin 16 expression and downregulates mu-opiate receptor expression in human epidermis.

It has been reported that opioid peptides modulate the differentiation of normal human keratinocytes and that mu-opiate receptors are expressed in human epidermis. The regulation of keratinocyte differentiation is particularly important in psoriasis, and one of the markers for hyperproliferative and differentiating skin diseases is cytokeratin 16. The finding that the endogenous mu-opiate receptor ligand beta-endorphin is increased in serum of patients with psoriasis indicates that the mu-opiate system may play an important role in the pathophysiology of the skin. In this study, we addressed the question whether there is a link between mu-opiate receptor regulation and cytokeratin 16 expression in normal and psoriatic skin. Firstly, we demonstrate that beta-endorphin concentrations between 16 and 1000 nM significantly downregulate mu-opiate receptor expression in epidermis of cultured human skin after 48 h. Secondly, we show that beta-endorphin regulates cytokeratin 16 expression in the epidermis of skin organ cultures exposed to 41-125 nM beta-endorphin for 48 h, leading to elevated cytokeratin 16 production. As expected, the expression of cytokeratin 16 was detected primarily in the suprabasal layer. The same pattern was observed in psoriatic lesional skin, i.e., mu-opiate receptor expression was significantly downregulated and cytokeratin 16 expression upregulated. These results suggest that the mu-opiate receptor system and its ligand beta-endorphin are involved in the pathogenesis of psoriasis, especially in terms of differentiation.

Down-Regulation↗
PubMed2000

Preclinical comparison in AR4-2J tumor-bearing mice of four radiolabeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-somatostatin analogs for tumor diagnosis and internal radiotherapy.

Somatostatin analogs labeled with radionuclides are of considerable interest in nuclear oncology as diagnostic or therapeutic tools for somatostatin receptor (SSTR)-expressing tumors. We investigated the suitability of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as a replacement for the widely used diethylenetriaminepentaacetic acid, to enable stable labeling of somatostatin analogs with both therapeutic (90Y) and diagnostic (111In) radionuclides. The three clinically relevant somatostatin agonists, octreotide, vapreotide, and lanreotide, together with the newly designed Tyr3-octreotide (TyrOc), were conjugated to DOTA and labeled with 90Y or 111In. For all DOTA-somatostatin analogs tested, irrespective of the incorporated radionuclide, we observed favorable biodistribution profiles in AR4-2J tumor-bearing mice: 1) a rapid clearance from all SSTR-negative tissues except kidney; 2) a specific uptake in SSTR-positive tissues, including tumor; and 3) an excellent tumor penetration. The main route of excretion was via the kidneys. Nevertheless, DOTATOC was clearly superior to the other DOTA-somatostatin analogs tested, as well as OctreoScan, as indicated by the highest tumor-to-nontarget-tissue ratio, including the tumor-to-SSTR-positive-tissue ratios. The presence of different SSTR subtypes in the SSTR-positive tissues possibly contributes to these differential uptakes. We assume that the very favorable behavior of DOTATOC in our mouse model makes this radioligand very promising for future applications in nuclear oncology.

Animals↗
PubMed2000

Receptor targeting for tumor localisation and therapy with radiopeptides.

Receptor targeting with radiolabeled peptides has become very important in nuclear oncology in the past few years. The most frequently used peptides in the clinic are analogs of somatostatin (SRIF), e.g. OctreoScan, which contain chelators for the radioisotopes 111In, 86Y, 90Y, 67Ga, 68Ga and 64Cu or for 99mTc and 188Re. and were labelled with the halogens 123I and 18F. Radiolabeled analogs of &alpha-melanocyte-stimulating hormone (&alpha-MSH), neurotensin, vasoactive intestinal peptide (VIP), bombesin (BN), substance P (SP) and gastrin/cholecystokinin (CCK) are also being developed, evaluated in vitro and in vivo and tested for clinical application. This review focuses on the expression in tumors and the regulation of receptors for these neuropeptides as well as the development of novel chelator-peptide conjugates suitable for in vivo scintigraphy or internal radiotherapy. The state of the art of radiopeptide pharmaceuticals is illustrated with four SRIF analogs, modified with the macrocyclic chelator 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA): [D-Phe1]-octreotide (DOTAOC), [D-Phe1, Tyr3]-octreotide (DOTATOC), vapreotide (DOTAVAP) and lanreotide (DOTALAN). DOTA is almost a universal chelator capable of strongly encapsulating hard metals such as 111In and 67Ga for Single Photon Emission Tomography (SPET), 68Ga, 86Y and 64Cu for Positron Emission Tomography (PET) as well as 90Y for receptor-mediated radionuclide therapy and radiolanthanides which exhibit different interesting decay schemes. From biodistribution studies in experimental animals and from clinical data it is concluded that DOTATOC is currently the most suitable SRIF radiopeptide with the best potential in the clinic.

Amino Acid Sequence↗
PubMed2000

Endocytosis and transcytosis of an immunoliposome-based brain drug delivery system.

Immunoliposomes conjugated with the OX26 monoclonal antibody to the rat transferrin receptor can be used for brain delivery of small molecules. In the present study the uptake of OX26-immunoliposomes by target cells as well as their transcytosis across the blood-brain barrier was investigated. Microscopy of RG2 rat glioma cells incubated with fluorescence labeled OX26-immunoliposomes revealed intracellular co-localization of liposomal cargo, the liposomal membrane bilayer and the OX26 monoclonal antibody. The distinct particulate staining pattern was indicative for accumulation of OX26-immunoliposomes within endosomal or lysosomal compartments. Prolonged incubations demonstrated endosomal release of the liposomal cargo propidium iodide to the cytoplasm. A maximum of 50% of propidium iodide was released from the endosomal compartment after 24 hours of incubation. Transcytosis was studied using an in vitro model of the blood-brain barrier consisting of immortalized RBE4 rat brain endothelial cells. OX26-immunoliposomes did permeate across the RBE4 cell monolayer and showed a permeability coefficient of P(app) = 1.6 x 10(-5) ml/s. Transport was inhibited at low temperature, by competition with free OX26 or by exchanging the OX26 monoclonal antibody for an unspecific isotype antibody. Transcytosis of OX26-immunolipsomes was confirmed in vivo by the brain perfusion and capillary depletion technique. OX26-immunoliposomes were detected within the post-vascular compartment of brain parenchyma (PS product = 2.4 microl/g/min.) and were not associated with the brain microvasculature.

Animals↗
PubMed2000

Expression of connective tissue growth factor (CTGF) mRNA in plaques of human anterior subcapsular cataracts and membranes of posterior capsule opacification.

PURPOSE: To investigate the correlation between connective tissue growth factor (CTGF) mRNA expression and immunohistochemical characteristics of anterior subcapsular cataract (ASC) formation as well as posterior capsule opacification (PCO) development (expression of type I collagen, alpha-smooth muscle actin and tenascin) under in vivo and under in vitro conditions in human and porcine lens epithelial cells. METHODS: CTGF mRNA expression was investigated using in situ hybridization and RT-PCR. Expression of type I collagen, alpha-smooth muscle actin and tenascin was detected by immunohistochemical staining. RESULTS: CTGF mRNA was expressed in human cataractous plaques of ASC and human PCO membranes, and appeared simultanously with the expression of type I collagen, alpha-smooth muscle actin and tenascin. CONCLUSION: The predominant expression of CTGF mRNA in human ASC and human PCO membranes suggests a significant role of CTGF in the pathological course of these ocular disorders.

Actins↗
PubMed1999

Differential regulation of somatostatin receptor type 2 (sst 2) expression in AR4-2J tumor cells implanted into mice during octreotide treatment.

Octreotide is a somatostatin analogue that is widely used for cancer therapy and tumor imaging. Its efficacy in tumors depends mainly on the expression of the somatostatin receptor type 2 (sst 2). Desensitization and down-regulation of sst 2 after agonist exposure can have important consequences for patients under ongoing octreotide therapy because it may induce temporary tumor unresponsiveness and impair sst 2-based tumor scintigraphy. Therefore, we have investigated the effect of octreotide on sst 2 expression in vitro, as well as in a tumor mouse model. In vitro, short exposure to octreotide induced rapid dose-dependent down-regulation of sst 2 in the rat pancreatic AR4-2J cell line. Within 0.5 h, 80% of sst 2 had disappeared from the cell surface. A total recovery required 24 h and was shown to depend on protein synthesis, but not on new sst 2 mRNA transcription, indicating that sst 2 was probably degraded during the down-regulation process. Similar results were obtained in vivo. On the other hand, long-term continuous release of octreotide for 7 days, as achieved with octreotide-containing osmotic minipumps, caused sst 2 up-regulation in vivo, but not in vitro. Furthermore, this up-regulation of sst 2 in tumor-bearing scid mice was shown to depend on constant exposure of the animals to octreotide, as it was not observed when octreotide was given discontinuously in two s.c. daily injections. These results demonstrate that the continuous release of a small amount of octreotide, which in cancer therapy may be achieved with long-acting release formulations of the peptide, can induce sst 2 up-regulation on cancer cells. This may improve the efficacy of both tumor imaging and long-term octreotide therapy.

Animals↗
PubMed1999

(D-(p-benzoylphenylalanine)13, tyrosine19)-melanin-concentrating hormone, a potent analogue for MCH receptor crosslinking.

A photoreactive analogue of human melanin-concentrating hormone was designed, [D-Bpa13,Tyr19-MCH, containing the D-enantiomer of photolabile p-benzoylphenylalanine (Bpa) in position 13 and tyrosine for radioiodination in position 19. The linear peptide was synthesized by the continuous-flow solid-phase methodology using Fmoc-strategy and PEG-PS resins, purified to homogeneity and cyclized by iodine oxidation. Radioiodination of [D-Bpa13,Tyr19]-MCH at its Tyr19 residue was carried out enzymatically using solid-phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed-phase mini-column and HPLC. Saturation binding analysis of [125I]-[D-Bpa13,Tyr19]-MCH with G4F-7 mouse melanoma cells gave a K(D) of 2.2+/-0.2 x 10(-10) mol/l and a B(max) of 1047+/-50 receptors/cell. Competition binding analysis showed that MCH and rANF(1-28) displace [125I]-[D-Bpa13,Tyr19]-MCH from the MCH binding sites on G4F-7 cells whereas alpha-MSH has no effect. Receptor crosslinking by UV-irradiation of G4F-7 cells in the presence of [125I]-[D-Bpa13,Tyr19]-MCH followed by SDS-polyacrylamide gel electrophoresis and autoradiography yielded a band of 45-50 kDa. Identical crosslinked bands were also detected in B16-F1 and G4F mouse melanoma cells, in RE and D10 human melanoma cells as well as in COS-7 cells. Weak staining was found in rat PC12 phaeochromocytoma and Chinese hamster ovary cells. No crosslinking was detected in human MP fibroblasts. These data demonstrate that [125I]-[D-Bpa13,Tyr19]-MCH is a versatile photocrosslinking analogue of MCH suitable to identify MCH receptors in different cells and tissues; the MCH receptor in these cells appears to have the size of a G protein-coupled receptor, most likely with a varying degree of glycosylation.

Amino Acid Sequence↗
PubMed1998

Melanin-concentrating hormone system in the brain of the lungfish Protopterus annectens.

The neurochemical anatomy of the lungfish brain is of particular interest, because many features in these animals might be representative of the common ancestor of land vertebrates. In the present study, we have investigated the localization and biochemical characteristics of melanin-concentrating hormone (MCH)-immunoreactive material in the central nervous system of the African lungfish, Protopterus annectens. The most prominent group of MCH-immunoreactive cell bodies was found in the dorsal hypothalamus. Additional groups of MCH-immunoreactive perikarya were detected in the telencephalon within the medial and dorsal pallium, the medial subpallium, and the ventral part of the lateral subpallium. Brightly immunofluorescent nerve fibers were seen in the anterior olfactory nucleus, the ventral part of the medial pallium, the medial subpallium, and the anterior preoptic area. In the diencephalon, the hypothalamus and the medial region of the dorsal thalamus exhibited a dense accumulation of fibers. MCH-immunoreactive fibers were also found in the tectum and the tegmentum of the mesencephalon and within the reticular formation of the rhombencephalon. In the pituitary, several small groups of cells of the intermediate lobe showed a bright fluorescence. Reversed-phase high-performance liquid chromatography (HPLC) analysis of diencephalon and pituitary extracts resolved a major MCH-immunoreactive peak that coeluted with synthetic salmon MCH. The distribution of MCH in the brain of P. annectens suggests that, in lungfishes, this peptide may exert neuromodulator or neurotransmitter functions. The presence of MCH-like immunoreactivity in the intermediate lobe of the pituitary indicates that, in dipnoans, MCH may also act as a typical pituitary hormone.

Animals↗
PubMed1998

Melanoma cell growth inhibition and melanocortin receptor downregulation induced by selective and non-selective retinoids.

The purpose of this study was to investigate the effects of retinoid analogues with different retinoid receptor specificity on the growth of human D10 and Cloudman S91 mouse melanoma cells. We compared the growth inhibitory effects with the ability of retinoids to downregulate cell surface expression of the melanocortin receptor (MC1-R). Retinoic acid receptor (RAR)-gamma-selective retinoids exerted the most prominent growth effects, with up to 68% and 69% inhibition in D10 and S91 cells, respectively. A retinoid X receptor (RXR)-selective compound inhibited cell growth by only 14% and 23% in D10 and S91 cells, respectively. Growth inhibition by RARalpha- and RARbeta-selective compounds was below 10% in both cells. In D10 cells, MC1-R downregulation was also induced most effectively by an RARgamma-selective retinoid (84% relative to controls). RARalpha-, RARbeta-and RXR-selective agonists induced only 16-24% MC1-R downregulation in these cells. The pattern for MC1-R downregulation was completely different in S91 cells. The RXR-selective compound was the most active (85%), followed by the RARalpha-selective agonist (58%), the RARgamma-selective compound (47%), and finally by the RARbeta-selective agonist (29%). We conclude that RARgamma-selective retinoids may have potential as therapeutic agents in melanoma. Different selectivity profiles for growth inhibition and MC1-R downregulation in S91 cells suggest that these two retinoid effects are not directly dependent on each other.

Animals↗
PubMed1997

Cloning of a human multispanning membrane protein cDNA: evidence for a new protein family.

We report the cloning of a human cDNA encoding a protein of calculated 68.8 kDa molecular mass, named hMP70. The deduced protein sequence shows a large N-terminal hydrophilic part and a C-terminal part with nine putative hydrophobic regions characteristic of integral transmembrane domains. Computer searches with sequence databases revealed homologies with three complete yeast proteins and with at least 19 human, 10 plant and one nematode short unidentified protein sequences translated from Expressed Sequence Tags (ESTs). Remarkably, this hMP70 protein retains between 27 and 31% overall sequence identity with the yeast proteins. We propose that hMP70 and related genes have evolved from a common ancestral gene and form a new multispanning membrane protein family which we call the MP70 protein family. Gene expression of hMP70 appears to be ubiquitous, as the mRNA is detectable in all human tissues analysed so far, as shown by Northern blot analysis. Furthermore, a protein of about 70 kDa is detectable in different mammalian cell lines, as shown by immunoblot analysis. From its widespread expression and conservation from yeast, plants to mammals, it is likely that hMP70 has a fundamental biological function in the cell.

Amino Acid Sequence↗
PubMed1997

Phosphorylation of the V2 vasopressin receptor.

The V2 vasopressin receptor undergoes ligand-induced sequestration and desensitization (Birnbaumer, M., Antaramian, A., Themmen, A. P. N., and Gilbert, S. (1992) J. Biol. Chem. 267, 11783-11788). The V2 receptor expressed in transfected cells labeled with [32P] orthophosphate was phosphorylated following the addition of 100 nM arginine vasopressin (AVP). Phosphorylation was complete 5 min after addition of AVP, and was not stimulated by increased levels of Ca2+ or cAMP. The half-maximal dose of AVP that stimulated phosphorylation was 2.4 +/- 0.4 nM, similar to the receptor KD of 4. 5 +/- 0.4 nM. The role of phosphorylation on receptor desensitization was investigated by studying two vasopressin receptors 14 and 27 amino acids shorter than the wild type receptor. The missing segments were not needed for normal ligand binding or coupling to Gs, but the last 14 amino acids were required for phosphorylation. The truncated receptors exposed to 100 nM AVP were sequestered and desensitized. The R137H V2R mutant receptor that binds vasopressin with wild type-like affinity and does not couple to Gs (Rosenthal, W., Antaramian, A., Gilbert, S., and Birnbaumer, M. (1993) J. Biol. Chem. 268, 13030-13033) was phosphorylated and subjected to ligand-induced sequestration. These results established that phosphorylation is not essential for sequestration and desensitization of the V2 vasopressin receptor. Furthermore, they revealed that the conformation acquired after ligand occupancy is necessary for receptor phosphorylation and sequestration, while coupling to Gs is not.

Adenylyl Cyclases↗
PubMed1997

Xanthines as a scaffold for molecular diversity.

Xanthines represent a new, versatile scaffold for combinatorial chemistry. A five-step solid-phase synthesis of xanthine derivatives is described which includes alkylations, a nucleophilic displacement reaction at a heterocycle and a ring closure reaction by condensation of a nitroso function with an activated methylene group. The selected reaction sequence allows the production of a highly diverse small-molecule combinatorial compound library.

Alkylation↗
PubMed1997

Identification of functional GRP-preferring bombesin receptors on human melanoma cells.

Bombesin was originally isolated from amphibian skin, whereas its mammalian counterpart, gastrin-releasing peptide (GRP), was first identified in the nervous system of the gastrointestinal tract. Whether GRP is present in the human skin is not known. Bombesin-like peptides are also known to modulate growth. We therefore investigated whether human melanoma cell lines express functional GRP-preferring bombesin receptors and whether they alter growth or other specific cellular functions of these tumour cells. GRP receptor mRNA was found in HBL, D-10, Me-28 and A375-6 cell lines, but only A375-6 cells express a large number of high-affinity binding sites for [125I]-[Tyr4] bombesin (K(d) 0.31 +/- 0.04 nmol L(-1), 3880 +/- 429 binding sites per cell). Bombesin dose-dependently increased cytosolic calcium, but did not alter interleukin (IL) 1beta-induced reduction of cell viability or IL-6 secretion, both A375-6-specific cell functions. Growth of A375-6 cells was not altered by bombesin or the specific GRP receptor antagonist BIM26226 as measured by [3H]-thymidine incorporation or methylene blue assay, whereas insulin alone or in combination with other potential growth factors dose-dependently stimulated growth of these cells. The newly characterized GRP-preferring bombesin receptors on highly malignant human melanoma cells could initiate studies of growth effects on solid tumours or in vivo scanning using radiolabelled tracers.

Bombesin↗