PubMed Health⌕ Search

Biomedical subjects

E Hintermann

Publications and source records attributed to E Hintermann.

13 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

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

Purification, cloning, and characterization of a second arylalkylamine N-acetyltransferase from Drosophila melanogaster.

In insects, amine acetylation by the enzyme arylalkylamine N-acetyltransferase (AANAT) is involved in melatonin formation, sclerotization, and neurotransmitter inactivation. This wide spectrum of activities suggests that several AANAT enzymes are present. We recently purified a protein fraction with AANAT activity from Drosophila melanogaster and cloned the corresponding gene, aaNAT1. Following the same strategy, we now report the purification of an additional AANAT from D. melanogaster, AANAT2, and the cloning of the corresponding cDNA. The isolated protein differs from AANAT1a and AANAT1b in its molecular weight and isoelectric point. The AANAT2 shares about 30% identity with the products of the aaNAT1 gene. The enzyme does not follow one-site Michaelis-Menten kinetics when assayed with various concentrations of the arylalkylamine tryptamine and a constant concentration (0.5 mM) of the cofactor acetyl coenzyme A. The data can be interpreted in terms of an enzyme with two kinetic regimes (K(m1) = 7.2 microM, K(m2) = 0.6 mM, and v(max2) = 2.7 v(max1)) that are governed by binding of the substrate to a regulatory site (K(r) = 6.2 mM). These findings demonstrate the presence of a second expressed gene encoding an AANAT in D. melanogaster. Northern blot analysis revealed no diurnal variation of aaNAT2 transcription, similar to the results obtained for aaNAT1a and aaNAT1b.

Acetylation↗
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

Molecular and biochemical characterization of the aaNAT1 (Dat) locus in Drosophila melanogaster: differential expression of two gene products.

In insects, arylalkylamine N-acetyltransferases (AANATs) have been implicated in several physiological processes, including sclerotization, inactivation of certain neurotransmitters, and, similar to the function in vertebrates, catalysis of the rate-limiting step in melatonin biosynthesis. Here, we report an extensive biochemical and functional analysis of the products of the aaNAT1 gene of Drosophila melanogaster. The aaNAT1 gene generates two transcripts through alternative first-exon usage. These transcripts are under tissue-specific and developmental control and encode proteins which differ in their N-terminus with respect to their starting methionine. The more abundant isoform, AANATlb, is first expressed during late embryogenesis in the brain, the ventral nerve cord, and the midgut; in adults, AANATlb is still detectable in the brain and midgut. The less abundant isoform, AANATla, appears only during late pupal stages and in adults is found predominantly in the brain. We demonstrate that the mutation Dat(lo) represents a hypomorphic allele of aaNAT1b, in which an insertion of two transposable elements, MDG412 and blastopia, has occurred within the first intron of the gene. Using a deficiency which removes the aaNAT1 gene, we provide evidence that aaNAT1 is not essential for the process of sclerotization. Furthermore, neither of the two enzyme isoforms shows circadian regulation of RNA or protein levels. The differing levels of abundance and distinct developmental control of AANAT1a and AANAT1b suggest different in vivo functions for these two enzymes.

Alternative Splicing↗
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↗
PubMed1996

Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut.

In insects, neurotransmitter catabolism, melatonin precursor formation, and sclerotization involve arylalkylamine N-acetyltransferase (aaNAT, EC 2.3.1.87) activity. It is not known if one or multiple aaNAT enzymes are responsible for these activities. We recently have purified an aaNAT from Drosophila melanogaster. Here, we report the cloning of the corresponding aaNAT cDNA (aaNAT1) that upon COS cell expression acetylates dopamine, tryptamine, and the immediate melatonin precursor serotonin. aaNAT1 represents a novel gene family unrelated to known acetyl-transferases, except in two weakly conserved amino acid motifs. In situ hybridization studies of aaNAT1 mRNA in embryos reveal hybridization signals in the brain, the ventral cord, the gut, and probably in oenocytes, indicating a broad tissue distribution of aaNAT1 transcripts. Moreover, in day/ night studies we demonstrate a diurnal rhythm of melatonin concentration without a clear-cut change in aaNAT1 mRNA levels. The data suggest that tissue-specific regulation of aaNAT1 may be associated with different enzymatic functions and do not exclude the possibility of additional aaNAT genes.

Acetylation↗
PubMed1995

Isolation and characterization of an arylalkylamine N-acetyltransferase from Drosophila melanogaster.

The enzyme arylalkylamine N-acetyltransferase (aaNAT) catalyzes the rate-limiting step in melatonin formation in the vertebrate pineal gland. Numerous attempts to purify this highly unstable enzyme from vertebrates have been unsuccessful. Here, we report the purification of an aaNAT enzyme from Drosophila melanogaster, using a radioenzymatic activity assay and column chromatography. The isolated 29.5-kDa protein acetylates tryptamine, dopamine and serotonin with affinities of 0.89 to 0.97 mM, respectively. This suggests that the identified aaNAT may be involved in melatonin synthesis and sclerotization as well as in neurotransmitter catabolism in insects.

Amino Acid Sequence↗
PubMed1995

Internal sequences from proteins digested in polyacrylamide gels.

A simple method for proteolytic digestion of Coomassie blue-stained proteins in a polyacrylamide matrix is presented. It consists of first reducing and alkylating the stained proteins with dithiothreitol and iodoacetamide in the presence of 0.1% sodium dodecyl sulfate and subsequent digestion with the endoproteinase LysC. The reduction and alkylation step was introduced since experiments with lysozyme and ribonuclease A showed that extremely complex peptide patterns were obtained if no precautions were taken to suppress disulfide bond formation during in-gel digestion of proteins. The advantage of this method is that no blotting step is required for generating internal sequences and that extensive proteolysis occurs which closely resembles that resulting from solution digests. The method has been successfully used to generate internal sequence data from low microgram quantities of proteins excised from 2-dimensional Coomassie blue-stained gels.

Acrylic Resins↗
PubMed1992

Bacteriophage T4 gene 21 encodes two proteins essential for phage maturation.

The T4 prohead protease (T4 PPase) is the key enzyme in the morphopoietic pathway of the T4 phage head. It is responsible for the proteolytic processing of all head proteins allowing protein rearrangement and head expansion. To study its biochemistry and gene regulation, T4 gene 21 was cloned into an expression vector under the control of the inducible tac promoter. Two proteins of apparent molecular weights of 21.5 and 27.5 kDa were detected after induction. These proteins are synthesized using two different start codons in the same reading frame. Destruction of either start codon resulted in the loss of the respective protein. Complementation experiments with bacteriophage T4 21(-)-infected cells showed that both proteins are functional in vivo and essential for T4 phage assembly.

Base Sequence↗
PubMedDate not supplied

Synthesis and characterization of new radioligands for the mammalian melanin-concentrating hormone (MCH) receptor.

Melanin-concentrating hormone (MCH) is a neuropeptide present in the brain of all vertebrates. For the characterization of MCH receptors, a monoiodinated [Phe13, Tyr19]-MCH radioligand analogue was developed. The high susceptibility of [125I]-[Phe13, Tyr19]-MCH to oxidative damage and its very lipophilic nature made it necessary to develop new MCH radioligands. To increase the stability, native methionines were replaced by non-sulphur containing amino acid residues. In one analogue, the L-enantiomer of the phenylalanine residue at position 13 was substituted by the D-enantiomer, which increased the relative affinity of the ensuing [125I]-[D-Phe13, Tyr19]-MCH about 7-fold. The different analogues were iodinated by an enzymatic reaction and used for binding studies with mouse melanoma cells. [125I]-[Met(O)4,8, Phe13, Tyr19]-MCH and [125I]-[Hse4,8, Phe13, Tyr19]-MCH showed only about 19% of total binding and [125I]-[Ser4,8, Phe13, Tyr19]-MCH displayed about 44% of total binding when compared with [125I]-[Phe13, Tyr19]-MCH. Non-specific binding for all tracers was below 11% of total binding of [125I]-[Phe13, Tyr19]-MCH binding. [125I]-[D-Phe13, Tyr19]-MCH was used for saturation binding studies and revealed a KD of 122.7 +/- 15.3 pmol/l. This radioligand was further characterized by association and dissociation binding studies.

Animals↗