Meta-iodobenzylguanidine (MIBG), a novel high-affinity substrate for cholera toxin that interferes with cellular mono(ADP-ribosylation).

Meta-iodobenzylguanidine (MIBG) is a guanidine analogue of the neurotransmitter norepinephrine. Radioiodinated [131I]MIBG is clinically used as a tumor-targeted radiopharmaceutical in the diagnosis and treatment of adrenergic tumors. Moreover, non-radiolabelled MIBG exerts several cell-biological effects, tentatively ascribed to interference with cellular mono(ADP-ribosyl) transferases (Smets, L.A., Bout, B. and Wisse, J. (1988) Cancer Chemother. Pharmacol. 21, 9-13; Smets, L.A., Metwally, E.A.G., Knol, E. and Martens, M. (1988) Leukemia Res. 12, 737-743). In the present study it was investigated whether MIBG could serve as an acceptor for the ribosyl transferase activity of cholera toxin and of erythrocyte membranes. MIBG appeared a substrate for the cholera toxin-catalyzed transfer of the ADP-ribose moiety of NAD to arginine-like residues with the highest affinity for this enzyme reported as yet (Km = 6.5 microM). MIBG was also ADP-ribosylated by the mono(ADP-ribosyl)transferase(s) of turkey erythrocyte membranes. Moreover, the drug appeared a potent affector of the ADP-ribose linkage to membrane proteins by these enzymes. Interference by MIBG was stronger than by related guanyltyramine, the monoamine precursors of MIBG, meta-iodobenzylamine had no effect at all. In contrast, the drug failed to affect endogenous, O-linked poly(ADP-ribose) polymerase, induced in nuclei of S49-leukemia cells by deoxyribonuclease. Since MIBG is the first described drug that specifically interferes with the cellular N-linked mono(ADP-ribosyl) transferase reactions, it may be an important tool to elucidate the physiological role of this posttranscriptional protein modification.

Intracellular inhibition of mono(ADP-ribosylation) by meta-iodobenzylguanidine: specificity, intracellular concentration and effects on glucocorticoid-mediated cell lysis.

meta-Iodobenzylguanidine (MIBG) is a high-affinity substrate for mono(ADP-ribosyl)transferase of cholera toxin and turkey erythrocyte membranes (Loesberg, C., Van Rooij, H. and Smets, L.A.(1990) Biochim. Biophys. Acta 1037, 92-99). In the present study the drug was investigated as a potential inhibitor of intracellular ribosyltransferases by competition with endogenous acceptors. To this end, MIBG was compared with the conventional ADP-ribosylation inhibitors nicotinamide and 3-aminobenzamide in cell-free ribosylation systems and in intact L1210 leukemia cells. Poly(ADP-ribose)polymerase (poly-ADPRP) was assayed by the DNAse-I-induced incorporation of [14C]NAD in nuclei of permeabilized L1210 cells. Mono(ADP-ribosyl)transferase (mono-ADPRT) was assayed as NAD linkage to [125I]iodoguanyltyramine catalysed by turkey erythrocyte membranes or activated cholera toxin. Poly-ADPRP was inhibited by nicotinamide (IC50 = 0.03 mM) and by 3-aminobenzamide (IC50 less than or equal to 0.03 mM) but was insensitive to MIBG. Conversely, mono-ADPRT was inhibited by MIBG (IC50 = approx. 0.1 mM) but not by 3-aminobenzamide and only weakly so by nicotinamide in high concentration (10 mM). In L1210 cells, intracellular levels of nicotinamide equilibrated at 60-70% of the extracellular drug concentrations assayed at 1 and 10 mM. In contrast, MIBG was concentrated 15-fold by nonspecific uptake. The preferential interference of the drugs with endogenous mono- or poly-ADP ribosylations, predicted from inhibitory capacity in vitro and intracellular concentrations, was confirmed by their effect on dexamethasone-induced lysis of L1210 cell lines. Inhibition of endogenous mono-ADPRT with 0.03 mM MIBG or 10 mM nicotinamide induced sensitivity to glucocorticoids in refractory L1210-wt cells. In contrast, inhibition of poly-ADPRP by 3-aminobenzamide or nicotinamide (1 mM each) did not confer susceptibility to refractory cells but enhanced the lytic process in the sensitive subline L1210-H7 or in L1210-wt cells sensitized by MIBG. These results indicate that MIBG is the first substrate for guanidino-specific mono-ADPRT which accumulates in intact mammalian cells and effectively competes with intracellular acceptors for endogenous enzymes.

Unusual specificity of the androgen receptor in the human prostate tumor cell line LNCaP: high affinity for progestagenic and estrogenic steroids.

UNLABELLED: LNCaP tumor cells, derived from a metastatic lesion of a human prostatic carcinoma, are androgen-sensitive in cell culture. Although increase in growth rate is observed with low doses of progestagens or estradiol, these cells contain exclusively androgen receptors. In the present study the binding affinity of different ligands for both non-DNA- and DNA-binding (transformed) forms of the androgen receptor were analyzed. The cytosolic (non-transformed) form of the receptor displayed an abnormal high affinity for progestagens and estradiol when compared with the cytosolic androgen receptor from other sources. Subsequently the non-transformed forms of the androgen receptor obtained from LNCaP cell nuclei was studied. A high binding affinity was found not only for dihydrotestosterone, but also for progesterone and the synthetic progestagen R5020 (relative binding affinity 42% and 10% of dihydrotestosterone). The binding characteristics of the transformed androgen receptor were examined in intact cells at 37 degrees C. LNCaP cells were compared in this respect with COS cells containing the cloned human androgen receptor, normal human skin fibroblasts and PC3 (prostate) and NHIK (cervix) human tumor cell lines. The affinity of the transformed androgen receptors for the progestagen R5020 in LNCaP cells was significantly higher than in the other cell systems, although the differences were less pronounced than for the non-transformed receptor form. IN CONCLUSION: the LNCaP tumor cells contain an androgen receptor with an abnormal binding site. This might be due to a mutation and/or a post-transcriptional effect.

Involvement of the glucocorticoid receptor in stress-induced apoptosis of leukemic cells.

Glucocorticoid (GC) hormones induce apoptosis in lymphoid and leukemic cells by binding and activating cytosolic GC receptors. Because physiological stress often causes hormone-free GC receptor activation, we have investigated if stress-induced apoptosis of lymphoid cells is also mediated by the activation of the GC receptor pathway. In S49 T lymphoma cells, heat shock and deprivation of growth factors or nutrients caused nuclear translocation and loss of agonist binding capacity of GC receptors, similar to that in cells incubated with the glucocorticoid dexamethasone (DEX). In variant S49 H.2 cells, cross-resistance to DEX, temperature shocks and growth factor deprivation were associated with a higher threshold for hormone-dependent and -independent receptor activation in situ and with impaired in vitro activation of cytosolic receptors. Cross-resistance to DEX, low serum and heat shock was abrogated, however, by pharmacological sensitization of GC receptor activation with the drug meta-iodobenzylguanidine (MIBG). Sensitive S49 cells and resistant variants did not differ in the expression levels of the apoptosis-regulating genes bax, bad, bcl-X and bcl-2, the status of the p53 gene nor in a different requirement for the growth factors II-2, IL-4 or IL-9. The results suggest that ligand-independent activation of the GC receptor is a central signalling and controlling event in some forms of stress-induced apoptosis, assigning a novel function to the GC receptor in the regulation of lymphoid and leukemic cell numbers.

Substitution of aspartic acid-686 by histidine or asparagine in the human androgen receptor leads to a functionally inactive protein with altered hormone-binding characteristics.

We have identified two different single nucleotide alterations in codon 686 (GAC; aspartic acid) in exon 4 of the human androgen receptor gene in three unrelated families with the complete form of androgen insensitivity. One mutation (G----C) results in an aspartic acid----histidine substitution (with 15-20% of wild-type androgen-binding capacity), whereas the other mutation (G----A) leads to an aspartic acid----asparagine substitution (with normal androgen-binding capacity, but a rapidly dissociating ligand-receptor complex). The mutations eliminate a Hinfl restriction site. Screening for the loss of the Hinfl site in both families with the Asp----Asn mutation resulted in the recognition of heterozygous carriers in successive generations of each. Both mutant androgen receptors were generated in vitro and transiently expressed in COS and HeLa cells. The receptor proteins produced had the same altered binding characteristics as those measured in fibroblasts from the affected subjects. R1881-activated transcription of a GRE-tk-CAT reporter gene construct was strongly diminished by both mutant receptors and was only partially restored using a 100-fold higher concentration of ligand compared with wild-type receptor. Thus, aspartic acid-686 appears essential for normal androgen receptor function. Substitution of this amino acid residue, by either histidine or asparagine, results in androgen insensitivity and lack of androgen-dependent male sexual differentiation.

An androgen response element in a far upstream enhancer region is essential for high, androgen-regulated activity of the prostate-specific antigen promoter.

Prostate-specific antigen (PSA) is expressed at a high level in the luminal epithelial cells of the prostate and is absent or expressed at very low levels in other tissues. PSA expression can be regulated by androgens. Previously, two functional androgen-response elements were identified in the proximal promoter of the PSA gene. To detect additional, more distal control elements, DNasel-hypersensitive sites (DHSs) upstream of the PSA gene were mapped in chromatin from the prostate-derived cell line LNCaP grown in the presence and absence of the synthetic androgen R1881. In a region 4.8 to 3.8 kb upstream of the transcription start site of the PSA gene, a cluster of three DHSs was detected. The middle DNAseI-hypersensitive site (DHSII, at approximately -4.2 kb) showed strong androgen responsiveness in LNCaP cells and was absent in chromatin from HeLa cells. Further analysis of the region encompassing DHSII provided evidence for the presence of a complex, androgen-responsive and cell-specific enhancer. In transient transfected LNCaP cells, PSA promoter constructs containing this upstream enhancer region showed approximately 3000-fold higher activity in the presence than in the absence of R1881. The core region of the enhancer could be mapped within a 440-bp fragment. The enhancer showed synergistic cooperation with the proximal PSA promoter and was found to be composed of at least three separate regulatory regions. In the center, a functionally active, high-affinity androgen receptor binding site (GGAACATATTGTATC) could be identified. Mutation of this element almost completely abolished PSA promoter activity. Transfection experiments in prostate and nonprostate cell lines showed largely LNCaP cell specificity of the upstream enhancer region, although some activity was found in the T47D mammary tumor cell line.

Density of beta adrenoceptors in rat heart and lymphocytes 48 hours and 7 days after acute myocardial infarction.

Down regulation of the beta adrenoceptor is thought to play an important role in the diminished response to catecholamines in heart failure. beta Adrenoceptor densities were measured on membrane homogenates of rat right ventricle and lymphocytes 48 h or 7 d after experimental myocardial infarction, and in rats exposed to a continuous infusion of isoprenaline (400 micrograms.kg-1.h1). The performance of the rat hearts was also evaluated 48 h post infarction in an isolated retrograde perfused heart preparation. In contrast to a 60% down regulation in right ventricle and a 20% down regulation in lymphocyte membranes after isoprenaline infusion, there was no change in right ventricle and lymphocyte beta adrenoceptor densities after myocardial infarction. Left ventricular contractile performance was significantly depressed 48 h after myocardial infarction. Mean basal left ventricular pressure decreased from 108(SEM 3) to 63(4) mm Hg while the maximal response to dobutamine was decreased from 204(4) to 105(12) mm Hg (n = 8). No correlation was found between the receptor densities of right ventricular and lymphocyte membranes. We conclude that diminished response to beta sympathomimetics after myocardial infarction cannot be attributed to a loss of surface beta adrenoceptors, and that the lymphocyte beta adrenoceptor does not provide an adequate system to monitor small receptor changes on the myocardium.

Mechanism of glucagon stimulation of fructose-1,6-bisphosphatase in rat hepatocytes. Involvement of a low-Mr activator.

Isolated rat hepatocytes were incubated in the absence or presence of glucagon and the activity of fructose-1,6-bisphosphatase was measured in cell extracts. After glucagon treatment the Vmax was increased (20-50%) whereas the Km remained unchanged. The stimulation was complete at 5 min after addition of glucagon. The glucagon concentration needed for maximal stimulation was 10(-9) M. After gel filtration the fructose-1,6-bisphosphatase activity in extracts of glucagon-treated cells was lowered to the control level. The effect of glucagon could not be completely mimicked by dibutyryl cAMP. The data indicate that in addition to the possible regulatory role of enzyme phosphorylation, a positive effector is involved in the stimulation of fructose-1,6-bisphosphatase activity by glucagon.

Structural organization of the human androgen receptor gene.

The complete coding region of the human androgen receptor gene has been isolated from a genomic library. The information for the androgen receptor was found to be divided over eight exons and the total length of the gene exceeded 90 kb. The sequence encoding the N-terminal region is present in one large exon. The two putative DNA-binding fingers are encoded separately by two small exons. The information for the hormone-binding domain is split over five exons. Positions of introns are identical to those reported for the chicken progesterone receptor and the human oestrogen receptor genes. Southern blot analysis of genomic DNA with various specific probes reveal that the human androgen receptor is encoded by a single-copy gene.

The effect of prednisolone and ketotifen on the antigen-induced bronchoconstriction and mediator release in rat isolated lungs.

Using a new method for inducing IgE-mediated, systemic anaphylaxis in the rat both prednisolone and ketotifen had been shown previously to be effective in suppressing the bronchial anaphylaxis in vivo. In order to study the mode of action underlying their bronchoprotective effect, both agents were also tested on the antigen-induced bronchoconstriction in rat isolated lungs in relation to the mediator release in the lung-effluent. The presence of histamine, 5-hydroxytryptamine (5-HT) and SRS-A could be detected biologically in the lung-effluent during bronchoconstriction. Histamine and 5-HT were determined quantitatively by means of h.p.l.c. with fluorimetric detection, whereas SRS-A was determined using the guinea-pig ileum in a cascade set-up. Although both prednisolone and ketotifen inhibited the antigen-induced bronchoconstriction effectively, it appeared that only prednisolone suppressed the release of histamine, 5-HT and SRS-A in the lung-effluent significantly, whereas ketotifen had no effect. On account of these data it is suggested that the bronchoprotective effect of prednisolone is mainly based on inhibition of the release of the mediators involved, whereas the effect of ketotifen may be based on receptor antagonism.

The N-terminal domain of the human androgen receptor is encoded by one, large exon.

Using specific cDNA hybridization probes, the first coding exon of the human androgen receptor gene was isolated from a genomic library. The exon contained an open reading frame of 1586 bp, encoding an androgen receptor amino-terminal region of 529 amino acids. The deduced amino acid sequence was characterized by the presence of several poly-amino acid stretches of which the long poly-glycine stretch (16 residues) and the poly-glutamine stretch (20 residues) were most prominent. Androgen receptor cDNAs from different sources contained information for poly-glycine stretches of variable size (23 and 27 residues, respectively). The androgen receptor amino-terminal domain was found to be hydrophilic and have a net negative charge. Combined with the previously described, partially overlapping cDNA clone 7A2M27 (Trapman et al. (1988) Biochem. Biophys. Res. Commun. 153, 241-248), the complete human androgen receptor was deduced to have a size of 910 amino acids.

Agonist-free transformation of the glucocorticoid receptor in human B-lymphoma cells.

Nuclear translocation of activated glucocorticoid receptors (GRs) is a necessary step in the signal transduction by these GC hormones. Although in vitro activation of GRs can occur in the absence of a functional ligand, it is generally assumed that binding of a cognate hormone is required for activation of the intracellular GR. By indirect immunocytochemistry and Western-blot analysis, it was found that, in spontaneously aggregated human lymphoma DoHH2 cells, hormone-free GRs are located in the nucleus. Disruption of the aggregates redistributed GRs to a predominantly cytosolic location. Upon spontaneous re-aggregation the GR again became localized to the nucleus. Intracellular cross-linking of the heteromeric receptor complex was applied to investigate the protein composition of cytoplasmic and nuclear receptors. Untransformed, cytosolic GRs could be demonstrated by [3H]dexamethasone binding capacity and hsp90 co-immunoprecipitation, whereas absence of these characteristics suggested an activated conformation of the nuclear GRs. These observations suggest that cell-cell interactions are capable of transforming GRs in the absence of a ligand.

High levels of non-activated receptors in glucocorticoid-sensitive S49wt mouse lymphoma cells incubated with dexamethasone.

Upon agonist binding the heteromeric glucocorticoid receptor complex undergoes a conformational change (receptor activation). This event involves the dissociation of a dimer of 90 kDa heat shock proteins. Whereas receptor activation in cytosolic assays is both rapid and irreversible, less is known about the receptor activation and translocation in intact cells during challenge with an agonist. In this paper we report on the receptor status of glucocorticoid-sensitive murine S49 lymphoma cells during dexamethasone exposure. By three different assays, ligand (re)binding, nuclear translocation and hsp90 co-immunoprecipitation, it was found that the majority of the glucocorticoid receptor protein was in a non-activated conformation. Furthermore, prolonged exposure to dexamethasone did not result in increased levels of activated receptors. By assessing receptor activation in situ we found that physiological temperature was less effective in dissociating hsp90 compared to room temperature. These findings indicate that the physiological temperature negatively controls receptor activation, probably due to a thermolabile interaction between the hormone and its cognate receptor.

Androgen receptor abnormalities.

The human androgen receptor is a member of the superfamily of steroid hormone receptors. Proper functioning of this protein is a prerequisite for normal male sexual differentiation and development. The cloning of the human androgen receptor cDNA and the elucidation of the genomic organization of the corresponding gene has enabled us to study androgen receptors in subjects with the clinical manifestation of androgen insensitivity and in a human prostate carcinoma cell line (LNCaP). Using PCR amplification, subcloning and sequencing of exons 2-8, we identified a G----T mutation in the androgen receptor gene of a subject with the complete form of androgen insensitivity, which inactivates the splice donor site at the exon 4/intron 4 boundary. This mutation causes the activation of a cryptic splice donor site in exon 4, which results in the deletion of 41 amino acids from the steroid binding domain. In two other independently arising cases we identified two different nucleotide alterations in codon 686 (GAC; aspartic acid) located in exon 4. One mutation (G----C) results in an aspartic acid----histidine substitution (with negligible androgen binding), whereas the other mutation (G----A) leads to an aspartic acid----asparagine substitution (normal androgen binding, but a rapidly dissociating androgen receptor complex). Sequence analysis of the androgen receptor in human LNCaP-cells (lymph node carcinoma of the prostate) revealed a point mutation (A----G) in codon 868 in exon 8 resulting in the substitution of threonine by alanine. This mutation is the cause of the altered steroid binding specificity of the LNCaP-cell androgen receptor. The functional consequences of the observed mutations with respect to protein expression, specific ligand binding and transcriptional activation, were established after transient expression of the mutant receptors in COS and HeLa cells. These findings illustrate that functional errors in the human androgen receptor have an enormous impact on phenotype and fertility.

Chemical characterization and comparative cellular effects of meta-iodobenzyl guanidine and benzyl guanidine.

meta-Iodobenzyl guanidine (MIBG) combines the structural properties of the neuron-blocking agents bretylium and guanethidine and is being used increasingly for various clinical applications. Different samples of MIBG were assayed for possible contamination with benzyl guanidine (BG). Fast-atom-bombardment mass spectrometry (FAB-MS) analysis showed a prominent but variable m/z 150 signal, corresponding to a protonated BG. The MS/MS fragmentation pattern of these [M + H]+ ions was similar to that obtained from FAB-MS-generated, protonated BG, confirming the proposed molecule and associated structures. RP-HPLC analysis of both guanidines, however, excluded the possibility of contamination of MIBG with BG. It was therefore concluded that the BG signal was an artifact of the FAB-MS procedure. In addition, the importance of the meta-substituted iodine for the biological activity of MIBG was investigated. Three different biochemical and cell-biological properties of MIBG were compared with those of its precursor MIBA and BG. The assays used were: inhibition of the catecholamine "Uptake I" system in SK-N-SH neuroblastoma and PC-12 pheochromocytoma cells, inhibition of mitochondrial respiration, and general cytotoxicity in L1210 leukemia cells. Of the drugs tested, MIBG was the most efficient in Uptake I inhibition and was more toxic in survival assays, but as compared with BG it was almost equipotent in inhibiting mitochondrial respiration. These findings contribute to a further elucidation of the mechanism by which MIBG exerts its various actions.

Cardiovascular responses to milrinone in pertussis toxin-pretreated pithed rats.

The modulating effects of pertussis toxin on angiotensin II and B-HT 920-evoked hemodynamic changes were compared with those of milrinone to evaluate the possible role of guanine nucleotide regulatory proteins (G proteins) in the mechanism of action of milrinone. Both milrinone and pertussis toxin shifted the blood pressure dose-response curves of B-HT 920 to the right, but the responses to angiotensin II were decreased after milrinone pretreatment only. The increase in cardiac frequency evoked by milrinone and isobutylmethylxanthine (IBMX) was not sensitive to pertussis toxin. In contrast, the decrease in systolic blood pressure elicited by milrinone could be prevented by pertussis toxin pretreatment, suggesting the involvement of a regulatory protein. Milrinone and IBMX did not influence the effects of arecoline on blood pressure or heart rate in either normal or pertussis toxin-pretreated rats. It is concluded that milrinone may affect a G protein, but not the adenylate cyclase-associated inhibitory protein, Gi.

The regional localization of R28935 in the cat brain as dependent on the route of administration.

Intravenous injection of the antihypertensive agent R2835 and its pharmacologically less active threo-isomer R29814 resulted in a distribution profile in the cat brain which differed from the regional localization after administration via the left vertebral artery. Although the two isomers had the same physico-chemical properties, R28935 penetrated more readily into the CNS. Intravenous administration resulted in almost equal levels in all brain parts, whereas after injection into the vertebral artery caudal structures contained more of both compounds that rostral structures. Differences existed between the concentrations in homotopic brain areas, especially in the brain stem. From comparison of the levels of R28935 after injection of an equiactive dose either i.v. or into the vertebral artery it is tempting to speculate that the mesencephalic tegmentum, the nucleus of the solitary tract, the inferior colliculi and/or the locus coeruleus are possible sites of the hypotensive action.

Plasma elimination of milrinone in rats in relation to its hemodynamic effects.

Milrinone (1,6-dihydro-2-methyl-6-oxo[3,4'-bipyridine]-5-carbonitrile) is a cardiotonic drug, currently under clinical investigation for the treatment of congestive heart failure. Its positive inotropic properties and its strong vasodilative action contribute to a favorable therapeutic effect. Plasma elimination of milrinone is generally by renal excretion of the unchanged drug. Since hemodynamic effects might influence the pharmacokinetics of renally eliminated drugs, we have investigated the plasma elimination of milrinone after different iv bolus doses of the compound in healthy rats. Half-times were 20.6 +/- 5.5 min (0.3 mg/kg milrinone), 17.0 +/- 1.8 min (1.0 mg/kg), 31.4 +/- 4.5 min (3.0 mg/kg), and 95.5 +/- 18.4 min (10.0 mg/kg). Clearances of milrinone showed a positive correlation with doses. Hemodynamic effects were dose dependent and (plasma) concentration dependent, and the maximum decrease in diastolic blood pressure was 54.5 +/- 0.5 mmHg. The half-effective plasma concentration of milrinone was 1.0 +/- 0.2 microM. The maximum increase in heart rate was 15%. We conclude that milrinone plasma elimination in rats is dose dependent, probably resulting from its strong vasodilator properties.

The combined use of high-performance liquid chromatography and radioimmunoassay for the bioanalysis of nicomorphine and its metabolites.

Methods have been developed for the determination of nicomorphine using reversed-phase HPLC with UV detection; for the simultaneous assay of morphine and mononicotinoylmorphine by a coupled normal-phase HPLC-radioimmunoassay method; and for conjugates of morphine and mononicotinoylmorphine by radioimmunoassay. The methods have been evaluated and applied to a pharmacokinetic study of nicomorphine administered intramuscularly.