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F Fahrenholz

Publications and source records attributed to F Fahrenholz.

At least 91 records · Page 5Linked to original sources
PubMed1990

Derivatives of somatic cell hybrids which carry the human gene locus for nephrogenic diabetes insipidus (NDI) express functional vasopressin renal V2-type receptors.

The gene responsible for familial vasopressin-resistant nephrogenic diabetes insipidus (NDI) has been localized to a small region of the human X-chromosome (Xq28). A series of hamster lung fibroblast and mouse lymphocyte cell lines carrying fragments of the wild type human X-chromosome was analyzed for vasopressin renal-type V2 receptor expression, to test the hypothesis that the NDI locus may have identity with the V2 receptor gene. V2 receptor binding activity and induction of cAMP production in response to [Arg8] vasopressin (AVP) were exhibited by all cell lines carrying the wild type NDI locus, in contrast to control cell lines. AVP stimulation of cAMP production was concentration-dependent and could be almost completely inhibited by co-incubation with a V2-V1 receptor-specific antagonist. The V2-specific agonist [Mpa1,Val4,Sar7]AVP was as potent as AVP in inducing cAMP production by NDI-DNA-carrying cells, whereas no response was shown to other hormones such as calcitonin, oxytocin (less than 10(-8) M), isoproterenol, or an oxytocin-specific agonist. All results were consistent with the hypothesis that the V2 receptor gene co-localized with the NDI locus, supporting the view that the loci are one and the same.

Animals↗
PubMed1990

Biotinyl analogues of vasopressin as biologically active probes for vasopressin receptor expression in cultured cells.

Biotinyl analogues of [Arg8]vasopressin were synthesized with the biotinyl moiety at position 4. This involved the substitution of 2, 4-diaminobutyric acid (Dab) for Gln4 in [1-deamino-Arg8]vasopressin to give the parent peptide des-[Dab4,Arg8]vasopressin. Two biotinyl analogues with different spacers between the side chain of Dab4 and the biotinyl residue were then prepared and characterized in detail. The analogues retained high binding affinities for the V2-receptor in both bovine kidney membranes and LLC-PK1 renal epithelial cells and for the V1-receptor in rat liver membranes. Both analogues were as potent as [Arg8] vasopressin in stimulating the cAMP-dependent protein kinase and the production of urokinase-type plasminogen activator in LLC-PK1 cells, with concentration dependence consistent with receptor binding affinities. Avidin or streptavidin did not appear to reduce receptor binding or biological activity of the biotinyl analogues. The use of the biotinylated vasopressin analogue des-[Dab-(biotinylamido)hexanoyl4, Arg8]vasopressin together with fluorescein-labeled streptavidin as a fluorescent probe for the V2-receptor in LLC-PK1 cells demonstrated the following: 1) Specific binding of the biotinyl analogue shown by quantitative single-cell fluorescence measurements using the technique of fluorescence microphotolysis; 2) the V2-receptor visualized by fluorescence microscopy; and 3) the expression of the V2-receptor detected by flow cytometry.

Amino Acid Sequence↗
PubMed1990

Monoclonal antibodies against different epitopes of peptide hormones. Use of photoreactive analogues in studies on vasopressin.

In order to produce monoclonal antibodies directed against different epitopes of the neurohypophyseal hormone vasopressin, the hormone was coupled to carrier proteins via photoreactive groups at different positions in the vasopressin sequence: [2-(4-azidophenylalanine), 8-arginine]vasopressin (peptide P1, photoreactive group at position 2) and desamino-[8-N6-(4-azidophenylamidino)lysine]vasopressin (peptide P2, photoreactive group at position 8) were conjugated to thyroglobulin by flash photolysis. Monoclonal antibodies against these conjugates bound ([3H]8-arginine]vasopressin with dissociation constants ranging over 40-400 nM. Epitope analysis by means of competitive ELISA showed that the monoclonal antibody obtained with peptide P1 as hapten was directed against the C-terminal acyclic tripeptide when its conformation was stabilized by interaction with the disulphide-linked cyclic hexapeptide. In contrast, the epitope analysis of three monoclonal anti-(peptide P2) antibodies demonstrated that they recognized antigenic determinants in the cyclic hexapeptide ring, mainly the hydrophobic surface formed by Tyr2 and Phe3. Our results suggest that monoclonal antibodies against different epitopes in small peptide hormones can be generated selectively by using photoreactive peptides in such a way that different antigenic sites are exposed in the hapten-carrier conjugate.

Amino Acid Sequence↗
PubMed1990

Lateral mobility of the phospholipase C-activating vasopressin V1-type receptor in A7r5 smooth muscle cells: a comparison with the adenylate cyclase-coupled V2-receptor.

The present work examines lateral mobility of the vasopressin V1-type receptor, representing the first determination of lateral mobility of a hormone receptor coupled to phospholipase C activation. The V1-receptor of A7r5 smooth muscle cells was characterized for [Arg8] vasopressin (AVP) binding properties and affinity for the fluorescent vasopressin analogue 1-deamino[8-lysine (N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP). TR-LVP was biologically active in A7r5 cells, inducing inositol 1,4,5-trisphosphate turnover in similar fashion to AVP. TR-LVP was used to specifically label the V1-receptor of living A7r5 cells, and lateral mobility of the V1-receptor was measured using the technique of fluorescence microphotolysis. The apparent lateral diffusion coefficient (D) at 37 degrees C was 5.1 x 10(-10) cm2/s, falling to 2.9 x 10(-10) cm2/s at 13 degrees C. These D values are higher than comparable values for the adenylate cyclase-activating vasopressin V2-receptor of LLC-PK1 renal epithelial cells analysed with the same fluorescent ligand. In contrast to the V2-receptor, no marked temperature dependence was observed for the V1-receptor mobile fraction (f). From 37 degrees C to 13 degrees C, f was relatively low (between 0.4 and 0.5) consistent with V1-receptor immobilization through internalization, which is rapid even at room temperature in A7r5 cells. These differences between V1- and V2-receptor lateral mobility are discussed in terms of the implications for their respective signal transduction systems.

Adenylyl Cyclases↗
PubMed1990

Ammonium chloride affects receptor number and lateral mobility of the vasopressin V2-type receptor in the plasma membrane of LLC-PK1 renal epithelial cells: role of the cytoskeleton.

The acidotropic agent ammonium chloride (NH4Cl) not only affects receptor metabolism by inhibiting lysosomal acidification, but can also affect the targeting of proteins to specific membranes in polarized cells, possibly through effects mediated by the cytoskeleton. The present study examines the effects of NH4Cl and perturbers of cytoskeleton structure on vasopressin V2 receptor expression in LLC-PK1 renal epithelial cells. Surprisingly, long-term pretreatment of cells with NH4Cl or short-term treatment with the actin perturber cytochalasin B resulted in an up to 70% increase in specific Arg-8-vasopressin binding compared to control cells, which was independent of the presence of NH4Cl in the binding test, and apparently the result of increased V2 receptor expression. Perturbers of microtubules such as colchicine and vinblastine had no such effect. A rhodamine-labeled analog of vasopressin was used to fluorescently label the V2 receptor of LLC-PK1 cells, and microscopic measurements of membrane-localized fluorescence confirmed the increased V2 receptor expression in the basal plasma membrane subsequent to NH4Cl pretreatment. Lateral mobility of the V2 receptor was measured in living cells using the technique of microphotolysis (photobleaching). The fraction of mobile receptors was 0.2 in cells pretreated with NH4Cl, markedly reduced compared to that of 0.9 in untreated cells. The apparent lateral diffusion coefficient D was about 3 x 10(-10) cm2/s in both pretreated and untreated cells. Results for fluorescence labeling of the actin cytoskeleton indicate that NH4Cl pretreatment of LLC-PK1 cells results in perturbation of microfilament structure. All results imply that the cytoskeleton plays a central role in V2 receptor expression and lateral mobility.

Actins↗
PubMed1990

Isolation of intact FNR protein (Mr 30,000) of Escherichia coli.

FNR, the activator of anaerobic respiratory genes of Escherichia coli, has previously only been isolated as a protein of Mr 29,000, which lacks nine N-terminal amino acid residues. The underlying proteolytic events have been studied with the aim of isolating intact FNR and determining whether cleavage is the result of a physiologically significant intracellular processing mechanism or proteolytic degradation during isolation. The FNR protein was present in aerobically and anaerobically grown bacteria as the intact protein (Mr 30,000). Proteolysis only occurred during and shortly after disruption of the bacteria. The production of FNR (Mr 29,000) must therefore be regarded as an isolation artefact. The proteolysis was caused by a protease which is located outside the cytoplasmic membrane or activated upon disruption of the membrane. Protease inhibitors directed against serine, cysteine or metalloproteases failed to prevent cleavage of FNR. In E. coli strain CAG627, proteolysis was greatly reduced making it possible to isolate FNR of Mr 30,000. The N-terminal sequence of FNR (Mr 30,000) was identical to that predicted from the fnr gene starting with the initiating methionine residue and including a four-cysteine cluster (16)Cys-X3-Cys-X2-Cys-X5-Cys(29).

Amino Acid Sequence↗
PubMed1990

A system to select for mutant LLC-PK1 cells affected in cAMP mediated hormonal response using a photoactivatable analogue of vasopressin.

The photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenyl-amidino)lysine] vasopressin (apa-LVP) could be used to elicit stimulation of cAMP production in LLC-PK renal epithelial cells, detectable up to 24 h after photoactivation by flash photolysis. This is in contrast to cells treated with vasopressin, or apa-LVP without photoactivation, where cAMP synthesis is down regulated within 4 h. The prolonged stimulation of cAMP production induced by photoactivation of apa-LVP was demonstrated to be cytotoxic to LLC-PK1 cells, whereas the vasopressin receptor negative LLC-PK1 mutant M18 was resistant to the cytotoxic effect. A selection strategy was developed for mutants resistant to this long-term stimulation of cAMP production, whereby multiple cycles of treatment with apa-LVP and photoactivation were used. Mutants so selected were then characterized using a novel screening system for detection of the production of urokinase-type plasminogen activator in response to cAMP agonists. One mutant was examined and found to be impaired in hormonal responsiveness, whereby hormone and forskolin stimulated cAMP-mediated responses were markedly reduced. It exhibited resistance to the long-term stimulation of cAMP production elicited by apa-LVP and photoactivation. This implies that apa-LVP can be used to select for novel mutants specifically impaired in cAMP metabolism and in particular down-regulation of cAMP response.

Adenylyl Cyclases↗
PubMed1989

Autoradiographic localization of V1 vasopressin binding sites in rat brain and kidney.

Monoiodination of the V1 vasopressin antagonist [Mca1,Sar7]AVP did not alter its high-affinity binding to liver plasma membranes. Monoradioiodinated [Mca1,125I-Tyr2,Sar7]AVP was therefore used to label V1-specific binding sites in the rat brain and kidney. The accumbens nucleus, the septal nucleus, the central amygdala, the bed nucleus of the stria terminalis, the stigmoid hypothalamic nucleus and the nucleus of the solitary tract exhibited specific labeling with both the radioiodinated V1 antagonist and tritiated AVP. Of the circumventricular structures only the choroid plexi and the area postrema showed V1-specific binding sites. The subfornical organ and hypothalamic loci of AVP synthesis such as the paraventricular nucleus, the supraoptic nucleus and the suprachiasmatic nucleus were not marked by the V1 antagonist while bearing [3H]AVP binding sites. As demonstrated by HPLC and binding to liver plasma membranes, the radiolabeled antagonist remained intact during tissue incubation. In addition to renal cortical and medullary [3H]AVP binding sites, medullary tubular and vascular structures could be labeled with the V1 antagonist, indicating the presence of both V1 and V2 AVP receptor subtypes in the rat kidney.

Amino Acid Sequence↗
PubMed1989

Major proteolytic fragments of the murine band 3 protein as obtained after in situ proteolysis.

Proteolytic fragments of murine band 3 were produced by exposure to extracellular chymotrypsin and intracellular trypsin. The ensuing proteolytic fragments were isolated, their N-terminal sequences were determined and their locations in the known amino acid sequence of murine band 3 established. Equivalents of the human 60, 35 and 17 kDa fragments were obtained through the cleavage sites were situated at locations that are not strictly homologous to the corresponding cleavage sites in human band 3, although all of them were near such sites. Exposure of the intact murine red cell to chymotrypsin leads to the formation of two fragments of 67 kDa and 41 kDa, which are equivalent to the 60 kDa and the 35 kDa fragments of the human band 3. Internal trypsin cleaves the chymotryptic 67 kDa fragment while the 41 kDa fragment appears essentially unaffected. The 67 kDa fragment is first degraded to 64 kDa, then further to 22 kDa and finally to 19 kDa. The anion transport inhibitor H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) combines with murine band 3 protein as it does with human band 3. Anion transport is maximally inhibited when 5.10(5) H2DIDS molecules per cell are bound to band 3. As in the human red cell, after exposure to high pH (9.0-9.5) of the H2DIDS-labeled, chymotryptically cleaved band 3 intramolecular cross-linking takes place. This joins the 67 and 41 kDa chymotryptic pieces together to form a peptide of the original molecular mass of band 3 of 108 kDa. If cross-linking is performed after additional tryptic cleavage, the 19 and 22 kDa pieces join together with 41 kDa pieces to form overlapping bands that cover the molecular weight range from 60 to 63 kDa.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Photoaffinity labelling of the oxytocin receptor in plasma membranes from rat mammary gland.

Plasma membranes from rat mammary gland containing a high concentration of [3H]oxytocin binding sites (2.8 pmol/mg protein) were used for photoaffinity labelling experiments. Competitive binding experiments show that these receptors bind with high affinity the specific oxytocin agonist [Thr4, Sar7]oxytocin and the analogue of 1-deamino-[8-lysine]vasopressin containing a photoreactive azidobenzoyl group (Abz) at the side chain of lysine. The tritium-labelled (50 Ci/mol) photoreactive analogue incorporated into a membrane protein with an apparent relative molecular mass of 65,000 +/- 3000 Da (n = 16). The labelling of this protein was completely suppressed by an excess of oxytocin.

Affinity Labels↗
PubMed1989

The adenylate cyclase-coupled vasopressin V2-receptor is highly laterally mobile in membranes of LLC-PK1 renal epithelial cells at physiological temperature.

The lateral mobility of membrane-associated hormone receptors has been proposed to play an important role in signal transduction. Direct measurements, however, have shown that the receptors for insulin, epidermal growth factor and beta-adrenergic antagonists exhibit low mobility at physiological temperature. The present study, which represents the first report of lateral mobility of a polypeptide hormone receptor coupled to adenylate cyclase, yielded quite different results. The lateral mobility of the vasopressin renal-type (V2)-receptor was measured in the basal plasma membrane of cells of the LLC-PK1 porcine epithelial line, using the technique of fluorescence microphotolysis (photobleaching) and a rhodamine-labelled analogue of vasopressin. The analogue, 1-deamino[8-lysine(N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP) was synthesized and shown to have binding properties and biological activities very similar to those of Arg8-vasopressin (AVP). TR-LVP could be used to label specifically the V2-receptor of living LLC-PK1 cells, whereby LLC-PK1 cells incubated with TR-LVP in the presence of a 100-fold excess of AVP, or cells from the LLC-PK1 V2-receptor-deficient line M18 incubated with TR-LVP could be used as controls for non-specific binding. Using optical sectioning, specific receptor mobility could be measured both in the absence and presence of free TR-LVP. The V2-receptor was found to be largely mobile at 37 degrees C: the mobile fraction (f) was approximately 0.9, and the apparent lateral diffusion coefficient (D) approximately 3.0 X 10(-10) cm2/s. V2-receptor mobility greatly decreased with decreasing temperature: at 10 degrees C f was reduced to approximately 0.1.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1988

Identification of a myometrial oxytocin-receptor protein.

The specific binding of [3H]oxytoxin to uterine membrane preparations derived from different species at late pregnancy was examined. The highest receptor density (bmax value) was found in membranes derived from the myometria of guinea pigs between day 60 post-conception (bmax = 3.6 +/- 0.1 pmol/mg) and day 65 (bmax = 4.4 +/- 0.1 pmol/mg). The similarity of Kd values for oxytocin binding (Kd = 2.6 +/- 0.2 nM) and for vasopressin binding (Kd = 2.1 +/- 0.4 nM) to the same membranes derived from a guinea pig myometrium indicate a homogeneous population of high-affinity binding sites which do not discriminate between these two hormones. Competitive binding experiments with specific oxytocin agonists containing either sarcosine or N-methylalanine in the place of Pro7 demonstrated that these myometrial receptors have the pharmacological properties of oxytocin receptors. The analogue of 1-deamino-[8-lysine]vasopressin containing a photoreactive azidophenylamidino group at the sidechain of Lys8 retained roughly the same receptor affinity as oxytocin. In photoaffinity labelling experiments with the tritium-labelled analogue a membrane protein from guinea pig myometrium with an apparent relative molecular mass Mr of 78,000 +/- 5000 (n = 13) was preferentially labelled. The labelling of this protein was completely suppressed by a 100-fold molar excess of either oxytocin, or [Sar7]oxytocin or [Thr4, Sar7]oxytocin, but not by other peptide hormones. These results provide evidence that the labelled 78,000-Mr protein is a myometrial oxytocin-receptor protein.

Affinity Labels↗
PubMed1988

Renal V2 vasopressin receptor proteins: identification and enrichment.

The synthesis of the tritium labelled photoreactive analogue of 1-deamino-vasopressin [1-(3-mercaptopropionic acid, 8-(N6-4-azido-phenylamidino)-lysine] vasopressin is described. This analogue retains a high affinity for hepatic V1 and renal V2 vasopressin receptors (apparent dissociation constant KD approximately 1-2 nM). A membrane protein from bovine kidney and pig kidney with an apparent relative molecular mass (Mr) of 30,000 was preferentially labelled and with lower yield a protein band with a Mr-value of 50,000 to 60,000. The photolabelled 30,000-Mr protein from bovine kidney was enriched by size-exclusion chromatography and by reversed-phase-high-performance liquid chromatography.

Affinity Labels↗
PubMed1987

Long-term stimulation of cAMP production in LLC-PK1 pig kidney epithelial cells by salmon calcitonin or a photoactivatable analogue of vasopressin.

A photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenylamidino)lysine]-vasopressin, was compared to salmon calcitonin and [8-arginine]-vasopressin with respect to stimulation of cAMP synthesis in the LLC-PK1 pig kidney epithelial cell line. Without photoactivation, the vasopressin analogue-elicited responses were identical to those induced by vasopressin, in that cAMP synthesis returned to the basal, unstimulated level about 4 h after hormonal treatment. In contrast, the levels of activation of cAMP-dependent protein kinase induced by salmon calcitonin returned to basal approx. 12 h after hormone addition. When activated by ultraviolet irradiation, the vasopressin analogue induced 'permanent' stimulation of adenylate cyclase, whereby cAMP production could be detected even 12.5 h after treatment. Both salmon calcitonin and the photoactivated vasopressin analogue inhibited growth of LLC-PK1 cells, in contrast to vasopressin or the nonactivated analogue. Growth inhibition appeared to be a consequence of the prolonged stimulation of adenylate cyclase. This conclusion was supported by the fact that a LLC-PK1 cell mutant in cAMP-dependent protein kinase was resistant to growth inhibition by salmon calcitonin and activated vasopressin analogue. The results imply that the cAMP-dependent protein kinase is the mediator of the hormone-stimulated growth inhibition.

Animals↗
PubMed1987

Synthesis and biological activities of a photoaffinity probe for vasotocin and oxytocin receptors.

The present study describes the synthesis and biological activities of the photoreactive vasotocin analog 1-deamino[8-lysine(N epsilon-4-azidobenzoyl)] vasotocin ([Mpa1, Lys(N epsilon-4-azidobenzoyl)8]vasotocin). The analog was obtained by introducing the photoreactive aryl azido group at the epsilon-amino group of Lys8 in [Mpa1, Lys8]-vasotocin, which was synthesized by the solid phase method. In the isolated toad urinary bladder the photoaffinity analog of vasotocin retained hydroosmotic activity in the absence of u.v.-light. After irradiation the osmotic water flow across the bladder wall increased. Moreover, the water permeability remained high during repeated periods of washout, suggesting that the analog formed covalent complexes with vasotocin receptors in the toad bladder. In the rat uterotonic assay the photoreactive vasotocin analog was without photoactivation a mild agonist. These studies suggest that the photoaffinity analog of vasotocin might be useful for the isolation of vasotocin receptors in low vertebrates and oxytocin receptors in mammals.

Animals↗
PubMed1986

Iodinated photoreactive vasopressin antagonists: labelling of hepatic vasopressin receptor subunits.

To identify and characterize V1 vasopressin receptors, photoreactive antagonists of the glycogenolytic and vasoconstrictor activity of vasopressin have been synthesized. The following analogues with 3-mercapto-3,3-cyclopentamethylene-propionic acid (Mca) and N-methylalanine (MeAla) in position 1 and 7 of vasopressin (VP) were effective V1 antagonists: [Mca1, D-Tyr2, MeAla7, Lys8]VP (1), [Mca1, MeAla7, Arg8, Lys9]VP (2), [Mca1, MeAla7, Arg8, D-Lys9]VP (3). Introduction of the photoreactive 4-azidophenylamidino group into the side-chain of Lys8 in analogue 1 or into Lys9 in analogues 2 and 3 increased the potency (for analogue 1 a tenfold increase in the antiglycogenolytic effect and a fivefold increase in the antivasopressor effect) and binding affinity for the rat hepatic V1 receptor. Mono-iodination at Tyr2 with 125I resulted in photoreactive antagonists of high specific radioactivity, which had roughly the same binding affinity as vasopressin for the rat hepatic V1 receptor (Kd = 0.9-1.8 nM). In photoaffinity labelling experiments with purified rat liver membranes, containing 2--3 pmol V1 receptor/mg protein, the analogues labelled specifically two proteins with the relative molecular masses (Mr) of 30,000 and 38,000. These results and the results of a recent study using 3H-labelled photoreactive vasopressin agonists [Boer, R. and Fahrenholz, F. (1985) J. Biol. Chem. 260, 15051-15054] provide evidence that both vasopressin agonists and antagonists can interact with the same two subunits of the heterodimeric hepatic V1 receptor. Furthermore the radioiodinated photoreactive V1 antagonists should be helpful to identify V1 receptor proteins in membranes of other cell types.

Affinity Labels↗
PubMed1986

Arginine-vasopressin analogues with high antidiuretic/vasopressor selectivity. Synthesis, biological activity, and receptor binding affinity of arginine-vasopressin analogues with substitutions in positions 1, 2, 4, 7, and 8.

In a search for more selective agonists of arginine-vasopressin (AVP), 10 analogues of [Sar7]- and [MeLa7]AVP with additional substitutions in positions 1 (beta-mercaptopropionic acid), 2 (phenylalanine), 4 (valine), or 8 (D-arginine) were synthesized and tested for antidiuretic and vasopressor activities. All analogues are characterized by a relatively high antidiuretic activity and by a sharp decrease in pressor activity. Their antidiuretic/vasopressor (A/P) selectivities were 2-3 orders higher (except for peptides 2 and 3) than that of the parent hormone. The additivity of the effects of changes in positions 1, 2, 4, and 8 combined with the sarcosine or N-methylalanine substitutions in position 7 on the biological activity is observed. Binding affinities of AVP analogues to plasma membranes from bovine kidney inner medulla and from rat liver containing specific vasopressin receptors were also determined. Generally, these analogues retained high binding affinities to renal vasopressin receptors, and on the other hand they are characterized by a large decrease in binding affinities to hepatic vasopressin receptors, which share characteristics with vasopressor receptors.

Alanine↗