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The human histamine H(4)-receptor (hH(4)R) is expressed in mast cells and eosinophils and mediates histamine (HA)-induced chemotaxis via G(i)-proteins. For a detailed investigation of hH(4)R/G(i)-protein interaction, we coexpressed the hH(4)R with Galpha(i2) and Gbeta(1)gamma(2) as well as an hH(4)R-Galpha(i2) fusion protein with Gbeta(1)gamma(2) in Sf9 insect cells. The agonist radioligand [(3)H]HA showed a K(D) value of approximately 10 nM at hH(4)R and hH(4)R-Galpha(i2). The high-affinity states of hH(4)R and hH(4)R-Galpha(i2) were insensitive to guanosine 5'-[gamma-thio]triphosphate (GTPgammaS). The affinity of [(3)H]HA for hH(4)R was retained in the absence of mammalian G(i)-proteins. In steady-state GTPase- and [(35)S]GTPgammaS-binding assays, hH(4)R exhibited high constitutive activity and uncommon insensitivity to Na(+). Thioperamide (THIO) was only a partial inverse agonist. Addition of HA or THIO to baculovirus-infected (hH(4)R + Galpha(i2) + Gbeta(1)gamma(2)) Sf9 cells increased the B(max) in [(3)H]HA binding, but not in immunoblots, suggesting conformational instability and ligand-induced stabilization of membrane-integrated hH(4)R. No effect was observed on hH(4)R-Galpha(i2) expression, neither in [(3)H]HA binding nor in immunoblot. However, the expression level of hH(4)R-Galpha(i2) was consistently higher compared to hH(4)R, suggesting chaperone-like or stabilizing effects of Galpha(i2) on hH(4)R. In 37 degrees C stability assays, HA stabilized hH(4)R, and THIO even restored misfolded [(3)H]HA binding sites. Inhibition of hH(4)R glycosylation by tunicamycin reduced the [(3)H]HA binding B(max) value. In conclusion, (i) hH(4)R shows high constitutive activity and structural instability; (ii) hH(4)R shows a G-protein-independent high-affinity state; (iii) hH(4)R conformation is stabilized by agonists, inverse agonists and G-proteins; (iv) hH(4)R glycosylation is essential for cell-surface expression of intact hH(4)R.

N1-Aryl(heteroaryl)alkyl-N2-[3-(1H-imidazol-4-yl)propyl]guanidines are potent histamine H2-receptor (H2R) agonists, but their applicability is compromised by the lack of oral bioavailability and CNS penetration. To improve pharmacokinetics, we introduced carbonyl instead of methylene adjacent to the guanidine moiety, decreasing the basicity of the novel H2R agonists by 4-5 orders of magnitude. Some acylguanidines with one phenyl ring were even more potent than their diaryl analogues. As demonstrated by HPLC-MS, the acylguanidines (bioisosteres of the alkylguanidines) were absorbed from the gut of mice and detected in brain. In GTPase assays using recombinant receptors, acylguanidines were more potent at the guinea pig than at the human H2R. At the hH1R and hH3R, the compounds were weak to moderate antagonists or partial agonists. Moreover, potent partial hH4R agonists were identified. Receptor subtype selectivity depends on the imidazolylpropylguanidine moiety (privileged structure), opening an avenue to distinct pharmacological tools including potent H4R agonists.

Doxorubicin (DOX), one of the most effective anticancer drugs, is known to generate progressive cardiac damage, which is due, in part, to DOX-induced reactive oxygen species (ROS). The elevated ROS often induce oxidative protein modifications that result in alteration of protein functions. This study demonstrates that the level of proteins adducted by 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product, is significantly increased in mouse heart mitochondria following DOX treatment. A redox proteomics method involving 2D electrophoresis followed by mass spectrometry and investigation of protein data bases identified several HNE-modified mitochondria proteins, which were verified by HNE-specific immunoprecipitation in cardiac mitochondria from the DOX-treated mice. The majority of the identified proteins are related to mitochondrial energy metabolism. These include proteins in the citric acid cycle (TCA) and electron transport chain (ETC). The enzymatic activities of the HNE-adducted proteins were significantly reduced in DOX-treated mice. Consistent with the decline in the function of the HNE adducted proteins, the respiratory function of cardiac mitochondria as determined by oxygen consumption rate (OCR) was also significantly reduced after DOX treatment. The treatment with Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnP, an SOD mimic, averted the doxorubicin-induced mitochondrial dysfunctions as well as the HNE protein adductions. Together, the results demonstrate that free radical-mediated alteration of energy metabolism is an important mechanism mediating DOX-induced cardiac injury suggesting that metabolic intervention may represent a novel approach to preventing cardiac injury after chemotherapy.

Mitochondrial dysfunction is a contributor to diabetic cardiomyopathy. Previously, we observed proteomic decrements within the inner mitochondrial membrane (IMM) and matrix of diabetic cardiac interfibrillar mitochondria (IFM) correlating with dysfunctional mitochondrial protein import. The goal of this study was to determine whether overexpression of mitochondria phospholipid hydroperoxide glutathione peroxidase 4 (mPHGPx), an antioxidant enzyme capable of scavenging membrane-associated lipid peroxides in the IMM, could reverse proteomic alterations, dysfunctional protein import, and ultimately, mitochondrial dysfunction associated with the diabetic heart. MPHGPx transgenic mice and controls were made diabetic by multiple low-dose streptozotocin injections and examined after 5 wk of hyperglycemia. Five weeks after hyperglycemia onset, in vivo analysis of cardiac contractile function revealed decreased ejection fraction and fractional shortening in diabetic hearts that was reversed with mPHGPx overexpression. MPHGPx overexpression increased electron transport chain function while attenuating hydrogen peroxide production and lipid peroxidation in diabetic mPHGPx IFM. MPHGPx overexpression lessened proteomic loss observed in diabetic IFM. Posttranslational modifications, including oxidations and deamidations, were attenuated in diabetic IFM with mPHGPx overexpression. Mitochondrial protein import dysfunction in diabetic IFM was reversed with mPHGPx overexpression correlating with protein import constituent preservation. Ingenuity Pathway Analyses indicated that oxidative phosphorylation, tricarboxylic acid cycle, and fatty acid oxidation processes most influenced in diabetic IFM were preserved by mPHGPx overexpression. Specific mitochondrial networks preserved included complex I and II, mitochondrial ultrastructure, and mitochondrial protein import. These results indicate that mPHGPx overexpression can preserve the mitochondrial proteome and provide cardioprotective benefits to the diabetic heart.

The human histamine H 3 receptor (hH 3 R) is a G-protein coupled receptor (GPCR), which modulates the release of various neurotransmitters in the central and peripheral nervous system and therefore is a potential target in the therapy of numerous diseases. Although ligands addressing this receptor are already known, the discovery of alternative lead structures represents an important goal in drug design. The goal of this work was to study the hH 3 R and its antagonists by means of molecular modelling tools. For this purpose, a strategy was pursued in which a homology model of the hH 3 R based on the crystal structure of bovine rhodopsin was generated and refined by molecular dynamics simulations in a dipalmitoylphosphatidylcholine (DPPC)/water membrane mimic before the resulting binding pocket was used for high-throughput docking using the program GOLD. Alternatively, a pharmacophore-based procedure was carried out where the alleged bioactive conformations of three different potent hH 3 R antagonists were used as templates for the generation of pharmacophore models. A pharmacophore-based screening was then carried out using the program Catalyst. Based upon a database of 418 validated hH 3 R antagonists both strategies could be validated in respect of their performance. Seven hits obtained during this screening procedure were commercially purchased, and experimentally tested in a [ 3 H]N a -methylhistamine binding assay. The compounds tested showed affinities at hH 3 R with K i values ranging from 0.079 to 6.3 lM.

The histamine H₄ receptor (H₄R) is expressed on cells of the immune system including eosinophils, dendritic cells, and T cells and plays an important role in the pathogenesis of bronchial asthma, atopic dermatitis, and pruritus. Analysis of the H₄R in these diseases depends on the use of animal models. However, there are substantial pharmacological differences between various H₄R species orthologs. The purpose of this study was to analyze the pharmacological properties of canine, rat, and murine H₄R in comparison to human H₄R expressed in Sf9 insect cells. Only hH₄R and cH₄R exhibited a sufficiently high [³H]histamine affinity for radioligand binding studies. Generally, cH₄R exhibited lower ligand-affinities than hH₄R. Similarly, in high-affinity GTPase studies, ligands were more potent at hH₄R than at other H₄R species orthologs. Unlike the other H₄R species orthologs, hH₄R exhibited high agonist-independent (constitutive) activity. Most strikingly, the prototypical H₄R antagonist (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) (JNJ7777120) exhibited partial agonistic activity at cH₄R, rH₄R, and mH₄R, whereas at hH₄R, JNJ7777120 was a partial inverse agonist. H₄R agonists from the class of N ( G )-acylated imidazolylpropylguanidines and cyanoguanidines exhibited substantial differences in terms of affinity, potency, and efficacy among H₄R species orthologs, too. The species-dependent pharmacological profiles are not due to the highly variable amino acid sequence position 341. Finally, H₄R species orthologs differ from each other in terms of regulation by NaCl. Collectively, there are profound pharmacological differences between H₄R species orthologs. Most importantly, caution must be exerted when interpreting pharmacological effects of "the prototypical H₄R antagonist" JNJ7777120 as H₄R antagonism.

It is assumed that many G protein-coupled receptors (GPCRs) are restrained in an inactive state by the "ionic lock," an interaction between an arginine in transmembrane domain (TM) 3 (R3.50) and a negatively charged residue in TM6 (D/E6.30). In the human histamine H 4 receptor (hH 4 R), alanine is present in position 6.30. To elucidate whether this mutation causes the high constitutive activity of hH 4 R, we aimed to reconstitute the ionic lock by constructing the A6.30E mutant. Thioperamide affinity at hH 4 R-R3.50A was increased by 300 to 400%, whereas histamine affinity was reduced by approximately 50%. A model of the active hH 4 R state in complex with the G␣ i2 C terminus was compared with the crystal structures of turkey ␤ 1 and human ␤ 2 adrenoceptors. We conclude that 1) constitutive activity of hH 4 R is facilitated by the salt bridge D5.69-R6.31 rather than by the missing ionic lock, 2) Y3.60 may form alternative locks in active and inactive GPCR states, 3) R3.50 is crucial for hH 4 R-G protein coupling, and 4) hH 4 R-R3.50A represents an inactive state with increased inverse agonist and reduced agonist affinity. Thus, the ionic lock, although stabilizing the inactive rhodopsin state, is not generally important for all class A GPCRs.According to the two-state model of receptor activation, GPCRs exist in an equilibrium between an active state (R*) and an inactive state (R), which are intrinsic properties of the receptor itself. R*, but possibly also R, couples to G proteins. R* promotes GDP/GTP exchange at the G␣ subunit and shows a higher affinity for agonists than R. Thus, agonists activate the receptor by stabilizing the R* state. The R* state is also stabilized by G proteins. Some receptors, e.g., hH 4 R (Schneider et al., 2009), human formyl peptide receptor 26 (FPR-26) (Wenzel-Seifert et al., 1998), or human ␤ 2 -adrenergic receptor (h␤ 2 AR) (Seifert et al., 1998) spontaneously adopt the R* state in the absence of agonists, which is referred to as constitutive activity. Inverse agonists show a preference for the R state and, therefore, reduce constitutive activity.Constitutive activity and inverse agonism are widespread phenomena in GPCR pharmacology and in many cases of pathophysiological importance (Seifert and Wenzel-Seifert, 2002). The hH 4 R shows very high constitutive activity and a G protein-independent high-affinity state, when coexpressed with mammalian G␣ i2 and G␤ 1 ␥ 2 in Sf9 insect cells (Schneider et al., 2009). Constitutive activity of the hH 4 R was not only observed in Sf9 insect cells, but also reported for mammalian cells, e.g., SK-N-MC cells, coexpressing hH 4 R with a