Label-free analysis of GPCR-stimulation: The critical impact of cell adhesion.

Label-free cell-based assays have been attracting growing attention in drug research. Optical approaches based on evanescent electric fields (e.g. EPIC, RWG/DMR, SPR) and electrochemical impedance analysis (ECIS, xCELLigence) are by far the most widespread techniques for such purposes. We compared three label-free approaches (ECIS, RWG/DMR and SPR) with respect to the activation of the human histamine H1 receptor (H1R) expressed by U-373 MG glioblastoma and genetically engineered HEK 293T cells. HEK 293T cells were either expressing the hH1R alone or in combination with the adhesion protein hMSR1. The ß2-adrenergic receptor (ß2-AR) expressed by bovine aortic endothelial cells (BAEC) served as a second cell model. Reduced cell adhesion to the surface of the sensing devices affected both, the optical and the impedance-based readout, but became much more obvious in case of RWG- or SPR-based assays. By contrast, the co-expression of hH1R and hMSR1 in HEK 293T cells strongly enhanced the signal compared to hH1R expression alone. As the sensitivity of the optical readouts is confined to a distance of 100-200nm from the surface, depending on the wavelength of the incident light, this observation is in accordance with tighter adhesion of the co-transfectants, inducing a shorter distance between the cell membrane and the substrate. Combining ECIS and SPR, allowing for simultaneous registration of both signals for a single cell population, provided a direct correlation of both readouts, when H1R or ß2-AR stimulation was investigated for the same cell populations. Cell adhesion was found to have a critical impact on the results of label-free cell monitoring, in particular when techniques based on evanescent electric fields are applied.

Label-free versus conventional cellular assays: Functional investigations on the human histamine H1 receptor.

A set of histamine H1 receptor (H1R) agonists and antagonists was characterized in functional assays, using dynamic mass redistribution (DMR), electric cell-substrate impedance sensing (ECIS) and various signaling pathway specific readouts (Fura-2 and aequorin calcium assays, arrestin recruitment (luciferase fragment complementation) assay, luciferase gene reporter assay). Data were gained from genetically engineered HEK293T cells and compared with reference data from GTPase assays and radioligand binding. Histamine and the other H1R agonists gave different assay-related pEC50 values, however, the order of potency was maintained. In the luciferase fragment complementation assay, the H1R preferred ß-arrestin2 over ß-arrestin1. The calcium and the impedimetric assay depended on Gq coupling of the H1R, as demonstrated by complete inhibition of the histamine-induced signals in the presence of the Gq inhibitor FR900359 (UBO-QIC). Whereas partial inhibition by FR900359 was observed in DMR and the gene reporter assay, pertussis toxin substantially decreased the response in DMR, but increased the luciferase signal, reflecting the contribution of both, Gq and Gi, to signaling in these assays. For antagonists, the results from DMR were essentially compatible with those from conventional readouts, whereas the impedance-based data revealed a trend towards higher pKb values. ECIS and calcium assays apparently only reflect Gq signaling, whereas DMR and gene reporter assays appear to integrate both, Gq and Gi mediated signaling. The results confirm the value of the label-free methods, DMR and ECIS, for the characterization of H1R ligands. Both noninvasive techniques are complementary to each other, but cannot fully replace reductionist signaling pathway focused assays.

Biodistribution of quantum dots in the kidney after intravenous injection.

The biodistribution of nanoparticles is a major subject of current nanomedical research. To date, however, the exact investigation of nanoparticle fate in the microenvironment of a main excretory organ, the kidney has largely been neglected. In this study, the biodistribution of polyethylene glycol-coated quantum dots (Qdots) with special focus on their interaction with the kidney is investigated. Upon intravenous injection, nanoparticles showed effective blood circulation in mice and significant renal accumulation after two hours. Histological analysis of the kidney revealed that Qdots were strongly associated to the intraglomerular mesangial cells. This preferential deposition of nanoparticles in the kidney mesangium is highly promising, since it could be of utmost value for site-specific treatment of severe kidney diseases like diabetic nephropathy in the future.

Increasing the throughput of label-free cell assays to study the activation of G-protein-coupled receptors by using a serial agonist exposure protocol.

Label-free, holistic assays, monitoring, for example, the impedance of cells on electrodes, are gaining increasing popularity in the evaluation of G-protein-coupled receptor (GPCR) ligands. It is the strength of these approaches to provide the integrated cellular response non-invasively, highly automated and with a device-dependent time resolution down to several milliseconds. With an increasing number of samples to be studied in parallel, the available time resolution is, however, reduced and the cost for the disposable sensor arrays may become limiting. Inspired by protocols from organ pharmacology, we investigated a simple serial agonist addition assay that circumvents these limitations in impedance-based cellular assays. Using a serial addition of increasing concentrations of a GPCR agonist while continuously monitoring the sample's impedance, we were able to establish a full concentration-response curve for the endogenous agonist histamine on a single layer of U-373 MG cells endogenously expressing the histamine 1 receptor (H1R). This approach is validated with respect to conventional, parallel agonist addition protocols and studies using H1R antagonists such as mepyramine. Applicability of the serial agonist addition assay was shown for other GPCRs known for their signaling via one of the canonical G-protein pathways, Gq, Gi/0 or Gs as well. The serial agonist addition protocol has the potential to further strengthen the output of label-free analysis of GPCR activation.

Stabilities of neutral and basic esters of bendamustine in plasma compared to the parent compound: kinetic investigations by HPLC.

Esters of the cytostatic bendamustine (1), previously demonstrated to be much more potent than the parent compound as antiproliferative agents in vitro, were investigated for stability in buffer and plasma, as well as against porcine liver esterase in the presence of different amounts of albumin using a validated RP-HPLC method with fluorescence detection. The hydrolysis of the nitrogen mustard moiety was retarded (for 1: approximately 130 vs. 11 min) in the presence of plasma proteins. For the derivatives, both cleavage of ester and nitrogen mustard moieties were analyzed. Enzymatic hydrolysis was very fast in the case of 2-pyrrolidino-, 2-piperidino- and 2-(4-methylpiperazino)-ethyl esters, whereas methyl, ethyl, morpholinoethyl and branched 2-pyrrolidinoethyl esters were considerably more stable (half-lives between 41 and 116 min, compared to <5 min). Inhibition by physostigmine indicated unspecific cholinesterases to be involved in the rapid ester cleavage. Due to lower protein content and higher enzymatic activity in murine compared to human plasma, reduced stability of all investigated esters in mouse plasma (t½<2 min) has to be taken into account with respect to the design of animal studies.

Molecular determinants for the high constitutive activity of the human histamine H4 receptor: functional studies on orthologues and mutants.

BACKGROUND AND PURPOSE: Some histamine H4 receptor ligands act as inverse agonists at the human H4 receptor (hH4 R), a receptor with exceptionally high constitutive activity, but as neutral antagonists or partial agonists at the constitutively inactive mouse H4 receptor (mH4 R) and rat H4 receptor (rH4 R). To study molecular determinants of constitutive activity, H4 receptor reciprocal mutants were constructed: single mutants: hH4 R-F169V, mH4 R-V171F, hH4 R-S179A, hH4 R-S179M; double mutants: hH4 R-F169V+S179A, hH4 R-F169V+S179M and mH4 R-V171F+M181S. EXPERIMENTAL APPROACH: Site-directed mutagenesis with pVL1392 plasmids containing hH4 or mH4 receptors were performed. Wild-type or mutant receptors were co-expressed with Gαi2 and Gß1 γ2 in Sf9 cells. Membranes were studied in saturation and competition binding assays ([(3) H]-histamine), and in functional [(35) S]-GTPγS assays with inverse, partial and full agonists of the hH4 receptor. KEY RESULTS: Constitutive activity decreased from the hH4 receptor via the hH4 R-F169V mutant to the hH4 R-F169V+S179A and hH4 R-F169V+S179M double mutants. F169 alone or in concert with S179 plays a major role in stabilizing a ligand-free active state of the hH4 receptor. Partial inverse hH4 receptor agonists like JNJ7777120 behaved as neutral antagonists or partial agonists at species orthologues with lower or no constitutive activity. Some partial and full hH4 receptor agonists showed decreased maximal effects and potencies at hH4 R-F169V and double mutants. However, the mutation of S179 in the hH4 receptor to M as in mH4 receptor or A as in rH4 receptor did not significantly reduce constitutive activity. CONCLUSIONS AND IMPLICATIONS: F169 and S179 are key amino acids for the high constitutive activity of hH4 receptors and may also be of relevance for other constitutively active GPCRs. LINKED ARTICLES: This article is part of a themed issue on Histamine Pharmacology Update published in volume 170 issue 1. To view the other articles in this issue visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2013.170.issue-1/issuetoc.

Synthesis and in vitro pharmacology of arpromidine and related phenyl(pyridylalkyl)guanidines, a potential new class of positive inotropic drugs.

Replacement of the cimetidine moiety in impromidine (1,N1-[3-(1H-imidazol-4-yl)propyl]-N2-[2-[[(5-methyl-1H-imidazol-4- yl)methyl]thio]ethyl]guanidine) by more lipophilic H2-nonspecific pheniramine-like structures resulted in potent H2 agonists with up to 160 times the activity of histamine in the isolated, spontaneously beating guinea pig right atrium. Additionally, the compounds proved to be moderate H1 antagonists. Highest H2-agonistic potency was found in compounds characterized by a three-membered carbon chain connecting the aromatic rings and the guanidine group. The activity in the atrium was increased 2-4-fold by halogen substituents in the meta or para position of the phenyl ring. Highest H1-antagonistic potency resides in the group of para-halogenated compounds, p-F representing the optimal substituent in both receptor models. The corresponding guanidine 52 (arpromidine, N1-[3-(4-fluorophenyl)-3-pyridin-2-ylpropyl]-N2-[3-(1H-imidazol-4- yl)propyl]guanidine) combines about 100 times the activity of histamine at the H2 receptor with H1-antagonistic potency in the range of pheniramine. Further increase in the activity on the atrium was achieved by disubstitution with halogen on the phenyl ring, such as 3,4-F2, 3,5-F2, and 3,4-Cl2 (63-65). The 2-pyridyl group in arpromidine was replaced by 3-pyridyl without significant change in H2 agonistic activity, whereas the 4-pyridyl and phenyl analogues were less active. The rank order of potency in the atrium was in good agreement with the positive inotropic effects found in isolated, perfused guinea pig hearts, where 63-65 were the most potent compounds as well.

Iodoaminopotentidine and related compounds: a new class of ligands with high affinity and selectivity for the histamine H2 receptor.

The synthesis and biological evaluation of a new class of histamine H2 antagonists with N-cyano-N'-[omega-[3-(1-piperidinylmethyl)phenoxy] alkyl]guanidine partial structure are described as part of an extensive research program to find model compounds for the development of new radioligands with high H2 affinity and specific activity. High receptor affinity is achieved by an additional (substituted) aromatic ring, which is connected with the third guanidine N by a carbon chain spacer and an amine, carboxamide, ester, or sulfonamide link ("polar group"). In functional studies for H2 antagonistic activity and other pharmacological actions [e.g. H1 antihistaminic, antimuscarinic, antiadrenergic (alpha 1, beta 1), 5-HT2 blocking activity] in the isolated guinea pig atrium and ileum and rat aorta and tail artery, the compounds proved to be highly potent and selective histamine H2 receptor antagonists. The H2 antagonistic activity is mainly depending on the length of both the N'-alkyl chain (chain A) and the N"-spacer (chain B). Compounds with a C3 chain A and a C2 chain B are most potent in the preferred group of substances, i.e., the carboxamide series. A wide variety of substituents at the aromatic ring is tolerated, among them iodine, amino, and azido groups. These compounds are up to 32 times more potent than cimetidine in the isolated guinea pig right atrium. The replacement of the carboxamide by an ester group (44c) is well tolerated, while replacement of the cyanoguanidine by an urea group results in nearly 100-fold decrease in activity (46c,e). The iodinated benzamides are among the most potent H2 antagonists known so far. The [125I]-labeled form of 31f ([125I]iodoaminopotentidine, [125I]-N-[2-(4-amino-3-iodobenzamido) ethyl]-N'-cyano-N"-[3-[3-(1-piperidinylmethyl) phenoxy]propyl]guanidine) and its photolabile analogue 31h ([125I]iodoazidopotentidine, [125I]-N-[2-(4-azido-3- iodobenzamido)ethyl]-N'-cyano-N"-[3-[3-(1-piperidinyl-methyl)pheno xy] propyl]guanidine) proved to be useful probes for reversible and irreversible labeling of the histamine H2 receptor. Radioligand binding studies in guinea pig cerebral membranes revealed considerably higher H2 receptor affinity for 31f (pKi = 9.15), 31h (pKi = 8.58), and some analogues than functional experiments (guinea pig atrium), presumably reflecting an easier access to the H2 receptors in membranes.

Pharmacokinetics and tissue distribution of bovine testicular hyaluronidase and vinblastine in mice: an attempt to optimize the mode of adjuvant hyaluronidase administration in cancer chemotherapy.

The influence of the route of administration (i.v., i.p. and s.c.) on pharmacokinetics and tissue distribution of bovine testicular hyaluronidase and vinblastine was studied in mice (plasma, skeletal muscle, liver, kidney and human melanoma). After i.v. injection, hyaluronidase was accumulated in liver and kidney, whereas i.p. and s.c. administration led to almost equal distribution in plasma, muscle, liver and kidney. In melanoma, the highest levels of hyaluronidase were found after s.c. injection of the enzyme close to the tumor. Hyaluronidase s.c. increased the intratumoral concentration of s.c. co-administered vinblastine most efficiently, making local simultaneous application as in interstitial chemotherapy most promising.

Quantitation of hyaluronidases by the Morgan-Elson reaction: comparison of the enzyme activities in the plasma of tumor patients and healthy volunteers.

The Morgan-Elson reaction, a method for the determination of hyaluronidase activity, was optimized for the quantitation of the enzyme in biological material. Based on HPLC and spectrometric (UV-Vis, LC-MS) studies, the structure of the red-colored product (mesomeric forms of N3-protonated 3-acetylimino-2-(4-dimethylaminophenyl)methylidene-5-(1,2-++ +dihydroxyethyl)furane) formed by condensation of chromogen III with p-dimethylaminobenzaldehyde is proposed. Activities corresponding to > or = 0.1 IU of endogenous and therapeutically administered hyaluronidase can be detected in 50 microl samples. Application of the method for the determination of the enzyme in plasma of tumor patients revealed no difference in activity levels, interindividual variability and pH profile compared to healthy volunteers.

Beneficial effect of H2-agonism and H1-antagonism in rat endotoxic shock.

Although histamine release is generally considered harmful in endotoxic shock, several data exist to doubt this view. Own previous studies in rats let us assume a possible beneficial effect only with H1-antagonists, however a detrimental effect on survival with H2-antagonists. Consequently H1- and H2-agonists and antagonists were studied to prove the hypothesis of a beneficial H2-agonistic and H1-antagonistic effect. Two randomized studies were performed in a standardized rat endotoxic shock model (45 mg of Escherichia coli endotoxin/kg body weight (b.w.)). In both, methylprednisolone (50 mg/kg b.w.) and saline were used as positive and negative controls, respectively. Study I compared the effects of H1- and H2-agonists (betahistine, .1 mg/kg/h, and impromidine, 100 micrograms/kg/h) with H1- and H2-antagonists (astemizole and famotidine both 1 mg/kg b.w.; 20 rats/dose). Study II was performed to estimate the dose-response relationship of a new, highly potent H2-agonist with additional H1-antagonistic features (BU-E 75: .01, .1, 1.0, 10, and 100 micrograms/kg/h; 20 rats/dose). Animals receiving impromidine or BU-E 75 all received omeprazole (1 mumol/kg b.w.) to suppress gastric acid secretion. In study I impromidine significantly increased the survival-time and -course compared to famotidine treated animals (p = .01 and p < .05). Study II showed a positive dose-response relationship of BU-E 75 with an increase in survival rates from 30% (.01 microgram/kg/h) to 70% (100 micrograms/kg/h). These data strongly support the hypothesis of a beneficial effect of H2-agonism and H1-antagonism on survival parameters in rat endotoxic shock.

Limited signal transduction repertoire of human Y(5) neuropeptide Y receptors expressed in HEC-1B cells.

In HEC-1B cells transfected with human Y(5) neuropeptide Y (NPY) receptors (but not in non-transfected cells) NPY inhibited forskolin-stimulated cAMP accumulation in a pertussis toxin-sensitive manner (-log EC(50) 8.88 +/- 0.25). Elevations of intracellular Ca(2+) were largely restricted to very high NPY concentrations and similar in transfected and nontransfected cells. NPY did not increase inositol phosphate accumulation and did not activate a variety of isoforms of protein kinase C or mitogen-activated protein kinases. We conclude that at least upon expression in HEC-1B cells the signal transduction of Y(5) NPY receptors is limited to inhibition of cAMP accumulation.

NPY Y1 antagonists: structure-activity relationships of arginine derivatives and hybrid compounds with arpromidine-like partial structures.

Previously, omega-guanidino- and omega-aminoalkanamides, structurally derived from arpromidine-like histamine H2 receptor agonists, were reported as novel neuropeptide Y Y1 antagonists. Regardless of the backbone, they resemble BIBP 3226, an argininamide with high NPY Y1 receptor affinity and selectivity, with respect to nature and arrangement of the 'terminal' diaryl, guanidine, and hydroxyphenyl groups. Hybrid compounds were synthesized combining the argininamide backbone of BIBP 3226 or partial structures derived from the C-terminal dipeptide of NPY with characteristic substructures of arpromidine- or amide-type NPY antagonists. Additionally, some analogs of BIBP 3226 with reduced flexibility were prepared. Structure-activity relationships indicate that, in contrast to alkanamides, homologs and/or isomers of BIBP 3226 with vicinal arrangement of the phenyl rings have decreased Y1 antagonistic activity (Ca2+-assay in HEL cells). Replacement of the hydroxybenzyl group by an imidazole ring further decreases activity. It is concluded that the binding sites of NPY antagonists with one and with two basic groups are not identical. Analogs with a rigid tetrahydro-2-benzazepine or an indan group in place of the benzyl moiety in BIBP 3226 are active, indicating the role of the OH group and supporting the model proposed for the interaction of BIBP 3226 with the Y1 receptor.

Agonist internalization by cloned Y1 neuropeptide Y (NPY) receptor in Chinese hamster ovary cells shows strong preference for NPY, endosome-linked entry and fast receptor recycling.

In Chinese hamster ovary (CHO) cells expressing the cloned guinea-pig Y1 receptor, the saturable, receptor-linked internalization of NPY (NPY)-related peptides showed the rank order of human/rat neuropeptide Y (hNPY)>pig/rat peptide YY (pPYY)>=(Pro(34))human PYY>(Leu(31),Pro(34))hNPY>(Leu(31),Pro(34))hPYY>>BVD-11 (a selective Y1 antagonist). All agonists accessed similar numbers of Y1 sites in particulates from disrupted cells, with relatively small affinity variation. The rate of internalization could significantly depend on the overall interactivity of the agonist peptide (reflected in sensitivity to chaotropic agents, as well as in the level of non-saturable binding and internalization). Concentration-dependent inhibition of the agonist-driven CHO-Y1 internalization was found with filipin III (a cholesterol-complexing macrolide), and confirmed with inhibitors of clathrin lattice formation, phenylarsine oxide (PAO) and sucrose. In the concentration range affecting Y1 internalization, none of the above treatments or agents significantly alter agonist affinity for Y1 cell surface or particulate receptors. Largely similar responses to the above inhibitors were observed in CHO-Y1 cells for internalization of human transferrin. Internalization of CHO-Y1 receptor apparently is driven by NPY in strong preference to other naturally encountered agonists. At 37 degrees C, most of the internalized receptor is rapidly recycled through endosome-like membrane elements, detectable in Percoll gradients.

Hyaluronidase pretreatment produces selective melphalan enrichment in malignant melanoma implanted in nude mice.

Preclinical and clinical observations suggest that the administration of hyaluronidase (Hyase) shortly before that of chemotherapy increases the access and, thus, the effectiveness of anticancer drugs in tumors. To examine this hypotheses as well as the selectivity of such a therapeutic approach potentially beneficial in isolated limb perfusion, the Hyase-induced distribution of melphalan was measured in tumor-bearing nude mice with respect to the mode of drug administration using RP-18 ion-pair high-performance liquid chromatography (HPLC) with fluorimetric detection. Melphalan alone (50 micromol/kg) or a combination of melphalan (50 micro mol/kg) and Hyase (100,000 IU/kg) was injected either i.p. or s.c. in the vicinity of the tumors. The s.c. melphalan injection caused a 4-fold rise in melphalan concentration (59 microM) in the tumors as compared with i.p. application (15 microM). Only minor effects were observed with respect to the route of melphalan application on its distribution in other tissues (ca. 13 microM in plasma, 15 microM in muscle, 30 microM in the liver, 26 microM in the kidney, and 21 microM in the testicle). Irrespective of the route of Hyase coadministration, the enzyme increased the concentration of i.p. injected melphalan in all tissues to ca. 20 microM in the tumor, 15 microM in plasma, 27 microM in muscle, 40 microM in the liver, 29 microM in the kidney, and 28 microM in the testicle. In contrast, s.c. injected melphalan was selectively accumulated by the tumors after both s.c. and i.p. Hyase administration (462 and 388 microM, respectively). Melphalan enrichment in the tumors was higher (16- to 32-fold higher than in the other tissues) after i.p. administration of Hyase since, in contrast to s.c. injection of the enzyme, its i.p. administration caused a decrease in the concentration of the cytostatic in all other tissues as compared with the s.c. administration of melphalan alone.

Histamine H1 receptors mediate vasodilation in guinea-pig ileum resistance vessels: characterization with computer-assisted videomicroscopy and new selective agonists.

Histamine receptors on guinea-pig ileum submucosal arterioles (outside diameter 40-80 microns) were studied in vitro using a computer-assisted videomicroscopy system (Diamtrak). Histamine receptor agonists investigated in this study were histamine, the H1 receptor-selective compound, 2-[2-(3-fluorophenyl)-4-imidazolyl]ethanamine (VZ 20), the H2 receptor-selective compounds, dimaprit, impromidine, (+/-)-N1-[3-(4-fluorophenyl)-3-(pyridin-2-yl)propyl]- N2-[3-(1H-imidazol-4-yl)propyl]guanidine (arpromidine) and (+/-)-N1-[3-(3,4-difluorophenyl)-3-(pyridin-2-yl)propyl]- N2-[3-(1H-imidazol-4-yl)propyl]guanidine (BU-E-75), as well as the H3 receptor-selective drug, (R)-alpha-methylhistamine ((R)-alpha-MeHA). Applied to vessels at resting tone, the agonists (1 nM-300 microM) did not change arteriolar diameter. Vessels preconstricted by 10 microM noradrenaline showed similar concentration-dependent vasodilations with histamine and VZ 20 (pD2 = 5.38 and 5.36, respectively). This histamine-induced vasodilation was not affected by tetrodotoxin (0.5 microM) or indomethacin (1 microM), but was completely abolished in the presence of 1 microM of the H1 receptor antagonist, mepyramine. Calculation of the antagonist affinity of mepyramine for the histamine receptors in submucosal arterioles yielded a pA2 of 9.46. In contrast to histamine and VZ 20, the H2 receptor agonist, dimaprit, and the H3 receptor agonist, (R)-alpha-MeHA, were ineffective at preconstricted arterioles. The guanidine-type H2 receptor agonists, impromidine, apromidine and BU-E-75, produced vasodilation at noradrenaline-preconstricted arterioles (-log EC50 = 4.47, 5.30 and 5.39, respectively) but, in contrast to histamine, were ineffective at arterioles preconstricted by U-46619 (300 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Programmable biodegradable implants.

Pulsatile release implants were developed that release substances up to 58 days post implantation. With a cylindrical size of 2 mm diameter and 1.8 mm height the matrices can carry as much as 1 mg of drug and allow even for intracranial implantation into a rodent model. The matrices are made of materials that have been used for parenteral applications in humans before such as surface eroding polyanhydrides and bulk eroding poly(D,L-lactic acid) or poly(D,L-lactic acid-co-glycolic acid). The onset of drug release is controlled by the degradation of bulk eroding polymers which are known to exhibit a certain stability over a defined period of time and which start eroding after they reach a critical degree of degradation. The time of drug release onset was found to depend on the molecular weight and the chemical state of the carboxylic acid end of the polymer chain. For testing the onset of release in vivo a nude mouse model was developed where the release of Evan's blue could be observed visually after subcutaneous application. By combining individual matrices with different release onset, a therapeutic system can be composed that releases drugs after implantation at predetermined time points in a preprogrammed way. Potential applications for such matrices is vaccination and local tumor therapy.

Characterization of histamine H2-receptors in human neutrophils with a series of guanidine analogues of impromidine. Are cell type-specific H2-receptors involved in the regulation of NADPH oxidase?

Human neutrophils possess an NADPH oxidase which catalyzes superoxide (O2-) formation and is activated by chemotactic peptides. Histamine inhibits O2- formation via H2-receptors (Burde et al. 1989). We characterized the neutrophil H2-receptor with a series of new guanidine-type H2-agonists structurally derived from impromidine. Histamine inhibited O2- formation with an IC50 value of 6.7 +/- 1.2 microM. Five aryloxy- and arylthioalkylguanidines were less potent and effective than histamine. Several arpromidine-like phenyl(pyridylalkyl)guanidines were either full or partial H2-agonists. Some guanidines possess a three-membered carbon chain connecting the aromatic rings and the guanidine group; they were similarly potent and effective as histamine. Shortening or elongation of the carbon chain substantially decreased the potency and intrinsic activity of the guanidines. Halogenation of the phenyl ring did not substantially affect the potency and intrinsic activity of the compounds in comparison to the non-substituted parent compound. The H2-antagonist, famotidine, competitively antagonized inhibition of O2- formation caused by the guanidine, arpromidine, with a pA2 value of 6.84. The H2-antagonist, cimetidine, differentially counteracted inhibition caused by partial and full H2-agonists. Partial H2-agonists antagonized the effects of histamine. The inhibitor of phosphodiesterases, 3-isobutyl-1-methylxanthine, additively enhanced the inhibitory effects of histamine and guanidines. The properties of the neutrophil H2-receptor were compared with literature data concerning properties of the H2-receptor of the guinea pig atrium. In the latter system, guanidines are full H2-agonists with potencies of up to 125-fold of that of histamine. Our data indicate that guanidines inhibit O2- formation in human neutrophils via H2-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Histamine inhibits activation of human neutrophils and HL-60 leukemic cells via H2-receptors.

The effects of prostaglandin E1 (PGE1) and histamine on activation of superoxide (O2-) formation, exocytosis of beta-glucuronidase and aggregation in human neutrophils and HL-60 leukemic cells were studied. PGE1, histamine and impromidine, a potent H2-agonist, inhibited O2- formation in neutrophils induced by the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe) with IC50 values of 0.5 microM, 8 microM and 2 microM, respectively. The full H1-agonist and weak partial H2-agonist, betahistine, was much less potent and effective than histamine. Dibutyryl cyclic AMP and forskolin mimicked the effects of histamine and PGE1 on O2- formation. The H2-antagonist, famotidine, competitively reversed histamine-induced inhibition of O2- formation with a pA2 value of 7.5. Histamine inhibited O2- formation when added prior to or after fMet-Leu-Phe. fMet-Leu-Phe-induced aggregation and release of beta-glucuronidase in neutrophils were less sensitive to inhibition by PGE1, histamine, dibutyryl cyclic AMP and forskolin than O2- formation. The inhibitor of cyclic AMP-specific phosphodiesterase, rac-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), additively enhanced the inhibitory effects of histamine and PGE1 on the above cell functions. In HL-60 cells differentiated by dimethyl sulfoxide or dibutyryl cyclic AMP, histamine, impromidine and PGE1 but not betahistine inhibited fMet-Leu-Phe-induced O2- formation as well. Our data suggest that histamine inhibits activation of neutrophils and HL-60 cells via H2-receptors through activation of adenylyl cyclase and increased formation of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Cationic-amphiphilic arpromidine-derived guanidines and a histamine trifluoromethyl-toluidide derivative may activate pertussis toxin-sensitive G-proteins by a receptor-independent mechanism.

Formyl peptides activate superoxide anion (O2-) formation in human neutrophils and in HL-60 cells via pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G-proteins), and histamine (HA) mediates inhibition of O2- formation via H2-receptors. We have studied the effects of lipophilic arpromidine-derived guanidines, which are potent, full H2-receptor agonists in the guinea pig atrium, on O2- formation and on activation of G-proteins in HL-60 membranes and on purified G-proteins. We have also studied the effects of a HA trifluoromethyl-toluidide derivative (HTMT), a cationic-amphiphilic HA derivative which activates O2- formation in HL-60 cells through a mechanism which is independent of known HA receptor subtypes, on G-protein activation. Guanidines, at concentrations, up to 30 mumol/l inhibited and, at concentrations above 30 mumol/l, enhanced formyl peptide-induce O2- formation in neutrophils. In HL-60 cells, guanidines per se activated O2- formation. The stimulatory effects of guanidines on O2- formation were not inhibited by H1- or H2-receptor antagonists. In HL-60 membranes, guanidines and HTMT, activated high-affinity GTPase in a PTX-sensitive manner. These substances also increased GTP hydrolysis effected by transducin and Gi/G(o)-proteins. Our data suggest that lipophilic guanidines and HTMT may act as receptor-independent activators of PTX-sensitive G-proteins, resulting in stimulation of O2- formation.