Histamine H1-receptors in HL-60 monocytes are coupled to Gi-proteins and pertussis toxin-insensitive G-proteins and mediate activation of Ca2+ influx without concomitant Ca2+ mobilization from intracellular stores

The results of binding studies suggest the presence of histamine H₁-receptors in human monocytes, but it is not known whether these receptors are functionally active. This prompted us to study the effects of histamine (HA) on cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and superoxide anion (O₂ ^– ) formation in HL-60 cells differentiated towards monocytes with 1,25-dihydroxychole-calciferol. In HL-60 monocytes, HA increased [Ca²⁺]_i with a half-maximal effect at 8 M and a maximum at 30–100 M. Pertussis toxin (PTX) partially inhibited the stimulatory effects of HA on [Ca²⁺]_i. Betahistine, a weak partial H₁-receptor agonist, also increased [Ca²⁺]_i, whereas H₂- and H₃-receptor agonists were ineffective. H₁- but not H₂- and H₃-receptor antagonists inhibited HA induced rises in [Ca²⁺]_i. HA-induced rises in [Ca²⁺]_i were desensitized in a homologous manner and were also inhibited by the activator of protein kinase C, 4\-phorbol 12-myristate 13-acetate. Various protein kinase C inhibitors did not interfere with homologous desensitization. The stimulatory effects of HA on [Ca²⁺]_i were completely dependent on the presence of extracellular Ca²⁺ and were inhibited by the blocker of non-selective cation (NSC) channels, 1-{\-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl}-1 H-imidazole hydrochloride (SK & F 96365). HA was much less effective than the chemotactic peptide, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP), to induce rises in [Ca²⁺]_i. Unlike fMLP, HA did not activate O₂ ^– formation. Our data indicate that HL-60 monocytes possess H₁-receptors coupled to heterotrimeric regulatory guanine nucleotide-binding proteins (G-proteins) of the G_i-family and PTX-insensitive G-proteins which mediate activation of NSC channels without concomitant activation of Ca²⁺ mobilization from intracellular stores, that homologous desensitization of HA-induced Ca²⁺ influx is independent of protein kinase C and that the stimulatory effect of HA on Ca²⁺ influx is too small to result in activation of O_{2 –} formation. Correspondence to: R. Seifert at the above address     

G-Protein-coupled receptors in HL-60 human leukemia cells

• 1.1. HL-60 human leukemia cells are a widely employed model system for the analysis of signal transduction processes mediated via regulatory heterotrimeric guanine nucleotide-binding proteins (G-proteins). HL-60 promyelocytes are pluripotent and can be differentiated into neutrophilic or monocytic cells.
• 2.2. HL-60 cells express formyl peptide-, complement C5a-, leukotriene B₄ (LTB₄)- and platelet-activating factor receptors, receptors for purine and pyrimidine nucleotides, histamine H₁- and H₂-receptors, β₂-adrenoceptors and prostaglandin receptors.
• 3.3. The major G-proteins in HL-60 cells are pertussis toxin (PTX)-sensitive G_i-proteins (G_i2 > G_i3). G_s-proteins and G-proteins of the G_q-family (e.g., G₁₆) are expressed, too.
• 4.4. G-protein-regulated effector systems in HL-60 cells are adenylyl cyclase and phospholipase C-β₂ (PLC-β₂) and, possibly, phospholipase D (PLD), nonselective cation (NSC) channels and NADPH oxidase.
• 5.5. The expression of signal transduction pathways in HL-60 cells strongly depends on the differentiation state of cells.
• 6.6. Formyl peptides, via G_i-proteins, mediate activation of PLC, PLD, NSC channels, NADPH oxidase and azurophilic granule release and are referred to as full secretagogues. In dibutyryl cAMP (Bt₂cAMP)-differentiated HL-60 cells, C5a and LTB₄ are partial and incomplete secretagogues, respectively. There are substantial differences in the G_i-protein activations induced by formyl peptides, C5a and LTB₄.
• 7.7. In HL-60 promyelocytes, purine and pyrimidine nucleotides mediate activation of PLC and NSC channels largely via PTX-insensitive G-proteins and induce functional differentiation. In Bt₂cAMP-differentiated HL-60 cells, they additionally activate PLD, NADPH oxidase and granule release via PTX-sensitive and -insensitive pathways. ATP and UTP are partial secretagogues. Multiple types of receptors (i.e., P_2Y- and P_2U-receptors and pyrimidinocyeptors) may mediate the effects of nucleotides in HL-60 cells.
• 8.8. Bt₂cAMP- and 1α,25-dihydroxycholecalciferol-differentiated HL-60 cells express H_l-receptors coupled to G_i-proteins and PTX-insensitive G-proteins. In the former cells, histamine mediates activation of PLC and NSC channels, and in the latter, activation of NSC channels. Histamine is an incomplete secretagogue in these cells.
• 9.9. HL-60 promyelocytes express H₂-receptors coupled to adenylyl cyclase, PLC, and NSC channels. There are substantial differences in the agonist/antagonist profiles of H₂-receptor-mediated cAMP formation and rises in cytosolic Ca²⁺ concentration, indicative of the involvement of different H₂-receptor subtypes. H₂-receptors mediate functional differentiation of HL-60 cells.
• 10.10. Certain cationic-amphiphilic histamine receptor ligands (i.e., 2-substituted histamines, lipophilic guanidines, and a histamine trifluoromethyl-toluidide derivative) show stimulatory effects in HL-60 cells that are attributable to receptor-independent activation of G_i-proteins.

     

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

Summary The effects of prostaglandin E₁ (PGE₁) and histamine on activation of superoxide (O_22/su–) formation, exocytosis of -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 µM, 8 µM and 2 µM, 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 -glucuronidase in neutrophils were less sensitive to inhibition by PGE₁, histamine, dibutyryl cyclic AMP and forskolin than_OZ formation. The inhibitor of cyclic AMP-specific phosphodiesterase, rac-4-(3-butoxy4-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. As stimulated basophils and mast cells release high quantities of histamine, this intercellular signal molecule may play an inhibitory role in the activation of cytotoxic functions of myeloid cells. Send offprint requests to R. Seifert at the above address     

Characterization of histamine H2-receptors in human neutrophils with a series of guanidine analogues of impromidine

Summary Human neutrophils possess an NADPH oxidase which catalyzes superoxide (O _inf2 ^sup– ) formation and is activated by chemotactic peptides. Histamine inhibits O _inf2 ^sup–1 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 O _inf2 ^sup– formation with an IC₅₀ value of 6.7 ± 1.2 M. 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 O _inf2 ^sup– 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-lmethylxanthine, 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 O _inf2 ^sup– formation in human neutrophils via H₂-receptors. The structure/activity relationship for the neutrophil H₂-receptor substantially differs from the one for the H₂-receptor in the guinea pig atrium, suggesting that the neutrophil H₂-receptor has cell type-specific properties. Other possibilities to explain the differences between H₂-receptors in these systems are discussed. Send offprint requests to R. Seifert at the above address     

Lack of effect of opioid peptides,morphine and naloxone on superoxide formation in human neutrophils and HL-60 leukemic cells

Summary There are controversial reports in the literature concerning the effects of opioids on superoxide (O ₂ ^– ) formation in phagocytes, these agents being either inhibitory or stimulatory. We re-examined this issue and compared the effects of the Chemotactic peptide, N-formyl-l,-methionyl-l-leucyl-l-phenylalanine (fMet-Leu-Phe), phorbol myristate acetate (PMA), ATP, platelet activating factor (PAF), cytochalasin B (CB) and prostaglandin E₁ (PGE₁) with those of various opioids on O ₂ ^– formation in human neutrophils and HL-60 leukemic cells under defined experimental conditions. In the presence of CB, fMet-Leu-Phe and PAF concentration-dependently activated O ₂ ^– formation in neutrophils with EC₅₀ values of 20 nM and 100 nM, respectively. In the absence of CB, fMet-Leu-Phe and PAF were much less effective. PAF synergistically enhanced O ₂ ^– formation induced by fMet-Leu-Phe. ATP at a concentration of 100 M and the opioids, methionine enkephalin, -endorphin, dynorphin, [d-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin, [d-Ala²-d-Leu⁵]-enkephalin and morphine at concentrations between 10 pM to 1 M did not activate O ₂ ^– formation. ATP but not \-endorphin potentiated fMet-Leu-Phe-induced O ₂ ^– formation. O ₂ ^– formation induced by a maximally stimulatory concentration of PMA (100 ng/ml) was enhanced by fMet-Leu-Phe but was unaffected by methionine enkephalin or PGE₁. PMA at a non-stimulatory concentration (2 ng/ml) potentiated the effect of fMet-Leu-Phe but did not induce responsiveness to PAF, ATP or -endorphin. PGE₁ strongly inhibited fMet-Leu-Phe-induced O ₂ ^– formation, whereas morphine, methionine enkephalin and the opioid antagonist, naloxone, were without effect. In HL-60 cells differentiated with dibutyryl cAMP, fMet-Leu-Phe, PAF and ATP but not -endorphin activated O ₂ ^– formation. Our results show that O ₂ ^– formation is differentially regulated by various classes of intercellular signal molecules and that opioids do not play a role in the regulation of O ₂ ^– formation. The precise definition of the experimental conditions and control experiments with established modulators of O ₂ ^– formation are essential to evaluate the role of opioids in the regulation of this effector system.Send offprint requests to R. Seifert at the above address     

Hydrogen peroxide modulates the Kv1.5 channel expressed in a mammalian cell line

Reactive oxygen species have been implicated in different types of cardiac arrhythmias including human atrial fibrillation. Kv1.5, the presumed molecular correlate of I_Kur, is an important determinant of human atrial repolarization. The aim of this study was to assess the effects of H₂O₂, at pathophysiologically relevant concentrations (20–1,000 M), on Kv1.5 expressed in Chinese hamster ovary cell line. Kv1.5 cDNA in pcDNA3 expression vector and CD8, a surface marker protein, were cotransfected in cells by calcium phosphate precipitation. Kv1.5 activation kinetics were significantly accelerated while the activation curve was negatively shifted by 10 mV (V_1/2 changed from –9.3 to –19.0 mV) in the presence of 100 M H₂O₂. The shift in Kv1.5 peak current I-V curve was voltage-dependent, the current amplitude being increased for voltages <+20 mV but decreased for high depolarizing voltages. The rapid activation time constant obtained from a bi-exponential fitting was decreased from 16.1±3.4 ms to 8.8±1.5 ms for a –20 mV depolarization (n=9; P=0.01) and from 4.3±2.1 ms to 2.3±0.4 ms when cells were depolarized to +20 mV (P<0.05). Kv1.5 steady-state inactivation was not modified by H₂O₂. Intracellular application of SOD or catalase reduced the H₂O₂ induced shift of activation I-V curve and SOD significantly decreased Kv1.5 amplitude at +40 mV (n=9; P<0.05). In conclusion, H₂O₂ increased Kv1.5 current amplitude at voltages corresponding to the action potential repolarization phase and accelerated Kv1.5 channel opening. These changes can reduce the action potential duration, leading to a shortening of the atrial effective refractory period. H₂O₂-induced changes in Kv1.5 properties could thus be involved in initiation or perpetuation of AF.David Caouette and Christiane Dongmo contributed equally to this article     

Differential effects of locally-applied capsaicin on distension-stimulated gastric acid secretion in the anesthetized rat

Summary The effects induced by the local administration of capsaicin on acid production have been investigated in the continuously perfused stomach of the anesthetized rat. Basal acid secretion was not influenced by 10 min intragastric perfusion with capsaicin (300 g min^–1). Acid responses elicited by distension of the stomach with increases in intragastric pressure of 5 and 10 cm H₂O were not modified after a 10 min intraluminal infusion with 80 or 300 g min^–1 of capsaicin. H⁺ output stimulated by higher intraluminal pressure (20 cm H₂O) were significantly decreased by intraluminal infusion of capsaicin (20, 80, 300 and 600 g min^–1). Acid responses to carbachol (4 g kg^–1, i.p.) were not influenced by intragastric (300 g min^–1), or systemic neonatal, treatment with capsaicin.Intraluminal infusion of the neurotoxin tetrodotoxin (0.12 g min^–1, 10 min) decreased acid responses to an increase in intragastric pressure of 20 cm H₂O but not those elicited by distention with a pressure of 10 cm H₂O. Neonatal systemic treatment (s. c.) with capsaicin or local gastric serosal application of either capsaicin or tetrodotoxin abolished acid responses to gastric distension (+ 20 cm H₂O). Capsaicin (80 g min^–1) and tetrodotoxin (0.12 g min^–1) infused concurrently into the lumen did not inhibit gastric acid secretion stimulated by an increase of 20 cm H₂O in intragastric pressure to any greater extent than did either drug given alone. The inhibition of acid secretion cannot be attributed to an increase in H⁺ loss, since intragastric infusion of capsaicin (300 g min^–1) did not cause any significant acid loss in rats pretreated with omeprazole and undergoing luminal perfusion with acid saline during gastric distension (+ 20 cm H₂O). Neither the intragastric perfusion nor the serosal application of capsaicin modified the H⁺ production induced by electrical stimulation of the vagus. This acid response was, however, abolished following 10 min intraluminal perfusion with tetrodotoxin (0.12 g min^–1).These observations confirm that capsaicin-sensitive sensory fibers located in the stomach wall mediate the acid secretory responses to intragastric distension. Such fibers are susceptible to the effects of capsaicin when administered systemically or through the gastric serosa, but only partially if capsaicin is infused intraluminally.Correspondence to: J. V. Esplugues     

In vivo continuous estimation of 3H-dopamine synthesis and release in the cat caudate nucleus

Summary Synthesis and release of ³H-DA were examined during the continuous superfusion of a limited area of the ventricular surface of the cat caudate nucleus with l-3,5-³H-tyrosine, using a cup technique. ³H–H₂O, an index of the conversion of l-3,5-³H-tyrosine into ³H-Dopa, and ²H-DA were estimated in serial superfusate fractions. Alpha-methyl-paratyrosine (-MpT) (10^–4 M) inhibited rapidly and simultaneously both ³H–H₂O formation and ³H-DA release. Transection of the nigro-striatal dopaminergic pathway immediately blocked ³H-DA release but ³H–H₂O levels were reduced by 30% one hour later.     

Changes in muscle blood flow after smoking a cigarette determined by a new noninvasive method

Summary The pharmacokinetics of the H₂-receptor antagonist famotidine, after oral administration of a 20 mg tablet, has been studied in 10 elderly patients with normal renal function (CL_CR59 ml·min^–1, Mean=80 ml·min^–1), 5 elderly patients with renal insufficiency (CL_CR38 ml·min^–1, Mean=15 ml·min^–1), and 6 healthy young volunteers.Elimination half-life in the elderly patients with renal insufficiency was significantly prolonged compared to the elderly patients with normal renal function and the young volunteers. The correlation coefficient between creatinine clearance and the elimination rate constant of famotidine was 0.672. Mean urinary recovery of unchanged drug up to 24 h in the young volunteers was 44%. The mean renal clearance of famotidine in the young volunteers (270 ml·min^–1) was substantially greater than the creatinine clearance, 128 ml·min^–1, which suggests the possibility of tubular secretion of famotidine.     

Long-term increase of hippocampal excitability by histamine and cyclic AMP

The action of histamine (HA) on rat hippocampal CA1 pyramidal cells in vitro was investigated in slices perfused with solution containing 0.2 mM Ca²⁺/4.0 mM Mg²⁺. Extracellular recordings of the spontaneous discharges occurring under these conditions revealed that HA caused a long-lasting increase in cell firing. The HA-effects were dose-dependent, in that low concentrations of HA (0.1–0.5 μM) exhibited an initial transient depression of cell firing and practically no long-lasting action, whereas higher concentrations of HA (1–10 μM) exerted strong, non-declining increases. The H₁-receptor antagonist mepyramine (1 μM) blocked the initial depression of firing and attenuated the long-lasting HA-mediated excitation. Pure H₁-receptor activation, tested with the H₁-receptor agonist 2-(3-fluorphenyl)histamine (1–10 μM) depressed cell firing, similar to the low dose effects of HA. HA-induced excitations were prevented by the H₂-receptor antagonist cimetidine (10–50 μM), and mimicked by the very potent H₂-receptor agonist impromidine (1 or 3 μM) which was, however, less effective compared to equal concentrations of HA. H₃-receptor activation by R-α-methylhistamine had no significant effect on cell firing. Thus, histamine H₁ and H₂ receptors seem to cooperate in producing this long-lasting augmentation of excitability. 8-Bromo-cyclic AMP monophosphate (8-Br-cAMP, 50–100 μM) mimicked the long-term excitation, whereas the adenylyl-cyclase inhibitor 9-tetrahydro-2-furyladenine (THFA, 100–500 μM) or the PKA-inhibitor Rp-adenosine-3′5′-cyclic monophosphate (Rp-cAMPS, 10 μM) blocked it, indicating that the HA-mediated increase of excitability in the hippocampus is dependent on the adenylate cyclase/PKA-signal transduction cascade. -2-Amino-5-phosphonopentanoic acid (APV, 50 μM) significantly attenuated the magnitude of the HA-induced enhancement, indicating an NMDA receptor-dependent component. Other biogenic amines, acting through receptors positively coupled to adenylyl cyclase, elicited similar responses as HA, indicating common mechanisms by which these substances modulate excitability in CA1 pyramidal cells.     

Hydrogen peroxide mobilizes Ca2+ through two distinct mechanisms in rat hepatocytes

Aim:

Hydrogen peroxide (H₂O₂) is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca²⁺ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes.

Methods:

Hepatocytes were extracted from rats. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i), inner mitochondrial membrane potentials and NAD(P)H levels were measured using fluorescence imaging. Phospholipase C (PLC) activity was detected using exogenous PIP₂. ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents.

Results:

H₂O₂ increased intracellular Ca²⁺ concentrations ([Ca²⁺]_i) across two kinetic phases. A low concentration (400 μmol/L) of H₂O₂ induced a sustained elevation of [Ca²⁺]_i that was reversed by removing extracellular Ca²⁺. H₂O₂ increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited H₂O₂-induced membrane current increases and [Ca²⁺]_i elevation. A high concentration (1 mmol/L) of H₂O₂ induced an additional transient elevation of [Ca²⁺]_i, which was abolished by the specific PLC blocker {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 but was not eliminated by removal of extracellular Ca²⁺. PLC activity was increased by 1 mmol/L H₂O₂ but not by 400 μmol/L H₂O₂.

Conclusion:

H₂O₂ mobilizes Ca²⁺ through two distinct mechanisms. In one, 400 μmol/L H₂O₂-induced sustained [Ca²⁺]_i elevation is mediated via a Ca²⁺ influx mechanism, under which H₂O₂ impairs mitochondrial function via oxidative stress, reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca²⁺ influx. In contrast, 1 mmol/L H₂O₂-induced transient elevation of [Ca²⁺]_i is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca²⁺ from intracellular Ca²⁺ stores.     

Relative Reactivity of Dihydropyridine Derivatives to Electrogenerated Superoxide Ion in DMSO Solutions: A Voltammetric Approach

Purpose. To evaluate the reaction of a large series of pharmacologically significant 1,4-dihydropyridine (1,4-DHP) compounds with superoxide (O^·– ₂) in dimethylsulfoxide using differential pulse voltammetry and controlled potential electrolysis. Methods. Differential pulse voltammetry was used to track the consumption of O^·– ₂, and controlled potential electrolysis was used to electrogenerate O^·– ₂. Results. With the addition of 1,4-DHP, the oxidation peak current of O^·– ₂ decreased concentration dependently, suggesting that 1,4-DHP reacts with O^·– ₂, that is, 1,4-DHP scavenges O^·– ₂ in dimethylsulfoxide. Conclusions. A very easy and direct voltammetric procedure to study the relative reactivity of different 1,4-DHP with O^·– ₂ is proposed. Using the proposed method we have found that all commercial 1,4-DHP reacts with O^·– ₂. The following order of rates was obtained: felodipine vitamin E > isradipine > nimodipine > furnidipine > nitrendipine > nisoldipine > nifedipine. Furthermore, it was demonstrated that the hydrogen at the N-position of 1,4-DHP compounds could be released as a proton in the presence of O^·– ₂, thus the electrogenerated O^·– ₂ worked as a proton acceptor to 1,4-DHP.     

Partial inhibition of human neutrophil activation by FK-506 at supratherapeutic concentrations

Summary The macrolide, FK-506, is a potent and effective inhibitor of lymphocyte activation. We studied the effects of FK-506 on human neutrophil activation induced by chemoattractants and by various substances which circumvent receptor stimulation. After preincubation for 5 min at 37°C, FK-506 (1 M) inhibited N-for-myl-L-methionyl-L-leucyl-L-phenylalanine (fMet-LeuPhe)-or platelet-activating factor-induced superoxide production in neutrophils by about 30%. At therapeutic concentrations (0.1–1 nM) FK-506 was ineffective. FK-506 did not inhibit exocytosis and rises in cytosolic Ca²⁺ concentration [Ca²⁺]_i mediated by these stimuli, and it did not at all inhibit neutrophil activation induced by C5a, leukotriene B4 and 4-phorbol 12-myristate 13-acetate. FK-506 (1 M) inhibited A23187-induced exocytosis by about 35%, but A23187-induced superoxide production was unaffected. After preincubation for 5 min at 37°C, FK-506 inhibited fMet-Leu-Phe-induced superoxide production in dibutyryl CAMP-differentiated HL60 cells by about 20%; preincubation with the drug for 24 h did not result in inhibition of superoxide production. FK-506 did not inhibit agonist-binding to formyl peptide receptors and fMet-Leu-Phe-stimulated GTP hydrolysis of heterotrimeric regulatory guanine nucleotide-binding proteins (G-proteins) in membranes from dibutyryl cAMP-differentiated HL,60 cells. FK-506 did not change steady-state and differential polarized phase fluorescence in HL-60 membranes using 1,6-diphenylhexa1,3,5-triene and 12-(9-anthroyloxy)-stearate as probes. Our results show that FK-506 at supratherapeutic concentrations partially inhibits neutrophil activation. Inhibition by FK-506 of fMet-Leu-Phe-induced superoxide production is rapid in onset and is not due to inhibition of agonist-binding to receptors, interference with G-proteins or protein kinase C, reduction of rises in [Ca²⁺]_i or alteration in physical membrane state.Correspondence to R. Seifert at the above address     

Identification of endothelial H1, vascular H2 and cardiac presynaptic H3 receptors in the pithed rat

Summary In pithed and vagotomized rats the effects of the H₃ receptor agonist R-(–)--methylhistamine, the H₁ receptor agonist 2-(2-thiazolyl)ethylamine and the H₂ receptor agonist dimaprit on basal diastolic blood pressure, basal heart rate and the electrically induced rise in heart rate were examined.Basal diastolic blood pressure was not altered by low, but increased by high doses of R-(–)--methylhistamine; the latter effect was not affected by selective H₁, H₂ or H₃ receptor antagonists and by prazosin, but was attenuated by rauwolscine. Rauwolscine also unmasked a vasodepressor response to R-(–)--methylhistamine not affected by the H₃ receptor antagonist thioperamide, but counteracted by the H₁ receptor antagonist dimetindene or the H₂ receptor antagonist ranitidine. The vasodepressor responses to 2-(2-thiazolyl)ethylamine and dimaprit were antagonized by dimetindene and ranitidine, respectively. The vasodepressor response to 2-(2-thiazolyl)ethylamine was not altered by indomethacin, but reduced by an inhibitor of endothelial nitric oxide synthase, N-nitro-L-arginine methyl ester (which, by itself, markedly increased blood pressure). Both drug tools did not alter the effect of dimaprit. Basal heart rate was not affected by 2-(2-thiazolyl)ethylamine (examined after administration of propranolol), dimaprit and R-(–)--methylhistamine. The electrically induced increase in heart rate (studied in animals which had received rauwolscine) was decreased by R-(–)--methylhistamine but not affected by 2-(2-thiazolyl)ethylamine and dimaprit. The effect of R-(–)--methylhistamine was abolished by thioperamide. R-(–)--methylhistamine did not influence the increase in heart rate produced by isoprenaline.In conclusion, the pithed rat offers the opportunity to study cardiac presynaptic H₃ receptors, endothelial H₁ receptors and vascular H₂ receptors in the same experimental model. Cardiac presynaptic H₁ and H₂ receptors as well as postsynaptic H₃ receptors in the heart and in the resistance vessels were not found. R-(–)--methylhistamine is a weak agonist at ₂, H₁ and H₂ receptors.Correspondence to E. Schlicker at the above address     

Mono- and divalent cations modulate the affinities of brain D1 and D2 receptors for dopamine by a mechanism independent of receptor coupling to guanyl nucleotide binding proteins

Summary In order to clarify the question of whether the modulatory effects of cations on dopamine receptor affinities are brought about by shifts in the equilibrium of receptor — G-protein — coupling, it was investigated whether mono- and divalent cations were still able to modulate rat striatal D1 and D2 receptor affinities after selective inactivation of the G-proteins linked to the two receptors. The G_S-protein coupled to the D1 receptor was eliminated by mild thermal inactivation, and the G_i- (or G_o-) protein associated with the D2 receptor by alkylation with a low concentration of N-ethyl-maleimide. Incubation of striatal membranes at 60°C completely abolished the specific binding of³H-GTP. Both treatments resulted in an increase of the IC₅₀-values for dopamine as a displacer of³H-SCH 23390 from D1- and of³H-spiperone from D2 receptors. Concomitantly, the formerly shallow D1 displacement curves became steeper, with their Hill coefficients increasing. This effect was less evident at D2 receptors. Guanosine triphosphate (GTP), which increased the IC₅₀'s of dopamine for both receptors approximately two-fold in control membranes, was without effect in pretreated samples, indicating an effective inactivation of the G-proteins. Na⁺ ions were still able to lower, and Ca²⁺ ions to increase the affinities of D1 and D2 receptors for dopamine after such inactivation of the respective G-proteins. It is concluded that the mechanism underlying the regulation of dopamine receptor affinities by mono- and divalent cations is independent of and superimposed upon the coupling of these receptors to guanyl nucleotide binding proteins.Abbreviations ANOVA Analysis of variance; G-proteins, guanyl nucleotide binding proteins (G_s: stimulatory, G_i: inhibitory); - GTP guanosine-5-triphosphate; Gpp(NH)p, 5-guanylylimidodiphos-phate; - NEM N-ethyl-maleimide     

Hexobarbital-binding,hydroxylation and hexobarbital-dependent hydrogen peroxide production in hepatic microsomes of guinea pig,rat and rabbit

Summary Cytochrome P-450 dependent oxygenase (3-hydroxy-hexobarbital) and oxidase activities (hydrogen peroxide) have been measured in hepatic microsomes from guinea pigs, rats and rabbits. A sensitive gas-chromatographic assay was developed to measure the hydroxylated product 3-hydroxy-hexobarbital. The kinetics of its formation were determined and correlated to hexobarbital type I binding and compared with oxidase activity: in the rat, V _max for 3-hydroxy-hexobarbital formation and for hexobarbital-dependent H₂O₂ formation was 5.1 and 2.6 nmoles/mg/min, resp. This was increased by phenobarbital tratment to 15.2 and 13.4 nmoles/mg/min. In phenobarbital treated rabbits, V _max was 15.0 nmoles/mg/min for hydroxylation and 40.8 for H₂O₂ formation. Spectral affinity constants (K _s) in control animals were 0.12 mM (rats) and 0.14 mM (rabbits). Phenobarbital treatment decreased these affinity constants, which were similar for each activity measured. In guinea pigs, however, hydroxylation of hexobarbital was low (3.1 nmoles/mg/min) and hexobarbital-dependent formation of H₂O₂ was higher than hydroxylation (V _max:7.0 nmoles/mg/min). Phenobarbital treatment led here to two affinity constants for each activity measured, which however, were alike. The existence of low in addition to high affinity constants observed here might explain the difficulties seen hitherto in correlating hexobarbital binding and metabolism in this species. Total oxidase activity was higher than oxygenase activity in all species tested.It is suggested that oxygenase activity of cytochrome P-450 is not limited by binding but by a competition with oxidase activity for a common intermediary species. This might be peroxy-P-450 (substrate-Fe³⁺O₂ ^2–), rendering either substrate-Fe³⁺O for hydroxylation reaction, or oxidized cytochrome P-450-substrate and hydrogen peroxide as product of oxidase function.Abbreviations S substrate - PB phenobarbital - HB hexobarbital - 3-OH-HB 3-hydroxy-hexobarbital - DPH diphenylhydantoin Supported in part by the Deutsche Forschungsgemeinschaft, Grant Hi 156/2, Biochemische Grundlagen der Arzneimittel- und Fremdstoffwirkungen     

β-ODAP accumulation could be related to low levels of superoxide anion and hydrogen peroxide in Lathyrus sativus L.

Level of the neuroexcitatory β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) in grass pea (Lathyrus sativus L.) varies with development and environmental stress. Reactive oxygen species (ROS) (mainly O₂⁻ and H₂O₂) are frequently reported to play important roles in plant development and in response to various stresses. To investigate the possible inter-relationship between contents of β-ODAP and ROS, grass pea leaves have been analyzed for contents of β-ODAP, O₂⁻ and H₂O₂. The results showed that leaves containing high levels of β-ODAP, exhibited low levels of O₂⁻ and H₂O₂, while leaves with high contents of O₂⁻ and H₂O₂ accumulated little β-ODAP. The application of pyridine or ABA which inhibit the production of O₂⁻ or H₂O₂ led to an increase in β-ODAP contents in intact or detached young leaves, whereas inhibition of catalase activity using AT (3-amino-1,2,4-triazole), leading to an increase in H₂O₂ content, result in significant decrease in β-ODAP levels of detached young leaves. In addition, inoculation of Rhizobium to young seedlings enhanced O₂⁻ and H₂O₂ levels, but reduced β-ODAP contents in shoots. These results suggest that β-ODAP accumulation could be related to low levels of superoxide anion and hydrogen peroxide in grass pea tissues.     

Augmentation of human neutrophil and alveolar macrophage LTB4 production by N-acetylcysteine: role of hydrogen peroxide

1. The actions of N-acetylcysteine (NAC) on hydrogen peroxide (H₂O₂) and leukotriene B₄ (LTB₄) production by human resting and stimulated peripheral blood neutrophils and alveolar macrophages were investigated.
2. At a concentration of 100 μM, NAC significantly (P<0.01) suppressed the accumulation of H₂O₂ in the incubation medium of resting and opsonized zymosan (OZ; 0.5 mg ml⁻¹)- or N-formylmethionyl-leucyl-phenylalanine (fMLP; 1 μM)-stimulated neutrophils and of resting and OZ-stimulated macrophages. At concentrations of 10 μM and above, NAC augmented significantly the level of LTB₄ in the supernatants of OZ- and fMLP-stimulated neutrophils (P<0.01 and P<0.05, respectively) and OZ-stimulated macrophages (P<0.05 at 10 μM, P<0.01 at 100 μM NAC).
3. NAC (100 μM) caused a significant (P<0.01) reduction in the quantity of measurable H₂O₂ when incubated with exogenous H₂O₂ concentrations equivalent to those released from OZ-stimulated neutrophils and macrophages. At no concentration did NAC affect quantitites of measurable LTB₄ when incubated with exogenous LTB₄.
4. Superoxide dismutase (SOD), which catalyzes the conversion of superoxide anion to H₂O₂ had no significant effect on LTB₄ production by human neutrophils. In contrast, catalase, which catalyzes the conversion of H₂O₂ to H₂O and O₂, caused a pronounced, statistically significant (P<0.01) increase in the levels of LTB₄ measured in the supernatants of OZ- and fMLP-stimulated neutrophils.
5. H₂O₂ (12.5 μM and 25 μM, concentrations equivalent to those measured in the supernatants of activated neutrophils and alveolar macrophages, respectively) caused a small (13%) decrease in the quantity of measurable LTB₄ (P=0.051 and P<0.05 at 12.5 μM and 25 μM, respectively) that was inhibited by NAC (100 μM) but not by catalase (400 u ml⁻¹).
6. In conclusion, the anti-oxidant drug, NAC, increases LTB₄ production by human neutrophils and alveolar macrophages, probably through the elimination of cell-derived H₂O₂. LTB₄ undergoes a H₂O₂-dependent oxidation that is inhibited by NAC but this is unlikely to account fully for the increased levels of LTB₄, suggesting that NAC may increase LTB₄ production by blocking the H₂O₂-dependent inhibition of a synthetic enzyme, such as 5-lipoxygenase.

     

[3H] GR67330, a very high affinity ligand for 5-HT3 receptors

Summary GR67330 potently inhibited 5-hydroxytryptamine (5-HT)-induced depolarizations of the rat isolated vagus nerve. At the higher concentrations used (0.3 nmol/l–1 nmol/l) this was accompanied by a marked reduction in the maximum response to 5-HT. The calculated pK_B value was 10.2.The binding of the tritiated derivative of GR67330 to homogenates of rat entorhinal cortex was examined. Kinetic analysis revealed that specific [³H] GR67330 (0.1 nmol/l) binding was rapid and reversible. Association and dissociation rate constants were 1.48 ± 0.36 × 10⁸ mol/l^–1 s^–1 and 7.85 ± 0.41 × 10^–3 s^–1 respectively. Equilibrium saturation analysis revealed specific binding was to a single site (Bmax 22.6±0.21 fmol/mg protein) of high affinity (Kd 0.038±0.003 nmol/l). At low ligand concentrations, specific binding was up to 90% of total binding. If unlabelled GR67330 was used to define non-specific binding two sites were evident (Kd₁ 0.066 ± 0.007 nmol/l, Kd₂ 20.1 ± 9.7 nmol/l; Bmax₂ 31.5 ± 3.2 fmol/mg protein, Bmax₂ 1110 ± 420 fmol/mg protein). [³H] GR67330 binding was inhibited potently by 5-HT₃ antagonists and agonists. Ligands for other 5-HT receptors and other neurotransmitter receptors were either only weakly active or inactive at inhibiting binding. Hill numbers for antagonist inhibition of binding were close to unity, except for quipazine which was significantly greater than one. In common with other 5-HT₃ binding studies, all 5-HT₃ agonist tested had Hill numbers greater than one (1.51–1.71). GR38032 and GR65630 inhibited a greater proportion of binding than other 5-HT₃ antagonists, this additional binding was interpreted as inhibition from a second saturable site unrelated to the 5-HT₃ receptor.Homogenates of five areas of rat brain were examined for specific [³H]-GR67330 binding (entorhinal cortex, cingulate cortex, parietal cortex, hippocampus and nucleus accumbens/olfactory tubercle). In each brain area a site of very high affinity was labelled. Drug inhibition profiles were also very similar in each brain area. It is concluded that, because of its high affinity, [³H] GR67330 will be a useful ligand to label 5-HT₃ receptors especially in tissues with low receptor densities and to map 5-HT₃ receptors autoradiographically.Abbreviations 5-HT 5-Hydroxytryptamine - 8-OH-DPAT 8-hydroxy-2-di-N-propylaminotetralin - 5-CT 5-carboxyamidotryptamine - GR38032 (±) 1,2,3,9-tetrahydro-9-methyl-3[(2-methyl-1H-imidazol-1-yl)methyl]-4H-carbazol-4-one - GR65630 3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl) -1-propanone - GR67330 (±)1,2,3,9 - tetrahydro-9-methyl-3-[(5-methyl-1H-imidazol-4-yl)methyl]-4H-carbazol-4-one - MDL72222 1H,3,5H-tropan-3-yl-3,5-dichlorobenzoate - ICS 205–930 (3-tropanyl)-1H-indole-3-carboxylic acid ester - BRL24924 endo-4-amino-5-chloro-2-methoxy-N-(1-azabicyclo[3,3,1]non-4-yl)benzamide - BRL43694 endo-N-(9-methyl-9-azabicyclo[3,3,1]non-3-yl)-1-methyl-indazole-3-carboxamide - SDZ 206-830 (3-homotropanyl)-1-methyl-5-fluoro-indole-3-carboxylic acid ester - mCPP meta-chlorophenylpiperazine Send offprint requests to G. J. Kilpatrick at the above address     

Immunomodulatory effect and quantitation of a cytotoxic glycosphingolipid from Murdannia loriformis

NMR signal reassignments for a cytotoxic glycosphingolipid compound, 2, -O-D-glucopyranosyl-2-(2-hydroxy-Z-6-enecosamide)sphingosine, isolated from an ethanolic extract of the herb Murdannia loriformis, have been achieved by use of FAB-MS, and 1D and 2D ¹H and ¹³C NMR. The amount of 2 in the herb juice was quantitatively determined by use of a validated HPLC method (RP-18, MeOH–H₂O, UV detection at 210 nm). The immunomodulatory effect of the herb juice and of 2 was proved by means of in vitro cellular immunological assays. Compound 2 at a concentration of 13 nmol L^–1 stimulated PBMC proliferation and increased the CD 3,4:CD 3,8 ratio in T lymphocytes.