β-Adrenergic Receptor Subtypes in Melanophores of the Marine Gobies Tridentiger trigonocephalus and Chasmichthys gulosus

The subtype of β-adrenergic receptors in melanophores of the marine gobies Tridentiger trigonocephalus and Chasmichthys gulosus was studied. Pigment of denervated melanophores in isolated, split caudal fins was preliminarily aggregated by incubating the specimens in a physiological saline containing 10 μM phentolamine and 30–100 μM verapamil or 2–10 nM melatonin, and the responses of the melanophores to a β-adrenergic agonist added to the incubating medium were recorded photoelectrically. The β-adrenergic agonists noradrenaline, adrenaline, isoproterenol, salbutamol and, dobutamine were all effective in evoking a dispersion of melanophore pigment in the presence of phentolamine and verapamil or melatonin. The pigment-dispersing effect of noradrenaline (β₁-selective agonist) was inhibited by metoprolol (β₁-selective antagonist), propranolol, and butoxamine. Whereas, the effect of salbutamol (β₂-selective agonist) was hardly inhibited by metoprolol, though it was considerably inhibited by propranolol and ICI-118551. It was estimated that β₁- and β₂-adrenergic receptors coexist at ratios of 8.6:91.4, in the melanophore of Tridentiger trigonocephalus, and 25:75, in the melanophore of Chasmichthys gulosus, through the analyses of Hofstee plots of the effects of the β-adrenergic drugs. It was suggested that the relation between the pigment-dispersing effect of a β-adrenergic agonist on the melanophores and the concentration of the drug follows mass action kinetics, when the effect is mainly caused by the activation of β₂-adrenergic receptors of the melanophores. However, when it is mainly caused by the activation of β₁-adrenergic receptors of the melanophores, the relation does not follow mass action kinetics.     

Melanophore pigment dispersion responses to agonists show two patterns of sensitivity to inhibitors of cAMP-dependent protein kinase and protein kinase C

Melanophore pigment dispersion is a sensitive bioassay for activation of the adenylyl cyclase and phospholipase C second-messenger pathways. The necessity of protein kinase activation in causing pigment dispersion was confirmed for eight agonists of endogenous melanophore receptors and for two transfected receptors. All agonists and receptors previously shown to elevate intracellular cAMP in melanophores—melanocyte stimulating hormone, light, (−) norepinephrine, 5-hydroxytrptamine, and the β₂-adrenergic receptor—were able to stimulate pigment dispersion in the presence of Ro31-8220, a potent inhibitor of protein kinase C, but were blocked in the presence of H89, an inhibitor of cAMP-dependent protein kinase. The bombesin receptor, which elevates intracellular IP₃ in melanophores, was unable to stimulate pigment dispersion in the presence of Ro31-8220 or H89. Agonists whose mechanism of activation of pigment dispersion are unknown were also tested. Endothelin 3 responses were blocked by both H89 and Ro31-8220, predicting coupling to phospholipase C. Vasoactive intestinal polypeptide, oxytocin, and calcitonin gene-related peptide β responses were blocked only by H89, predicting coupling to adenylyl cyclase. © 1996 Wiley-Liss, Inc.     

5-HT receptor subtypes as key targets in mediating pigment dispersion within melanophores of teleost,Oreochromis mossambicus

The presence of distinct class of 5-HT receptors in the melanophores of tilapia (Oreochromis mossambicus) is reported. The cellular responses to 5-HT (5-hydroxytryptamine), 5-HT₁, and 5-HT₂, agonists on isolated scale melanophores were observed with regard to pigment translocation within the cells. It was found that 5-HT exerted rapid and strong concentration dependent pigment granule dispersion within the melanophores. The threshold pharmacological dose of 5-HT that could elicit a measurable response was as low as 4.7 × 10^? 12 M/L. Selective 5-HT₁ and 5-HT₂ agonists, sumatriptan and myristicin were investigated and resulted in dose-dependent pigment dispersion. The dispersing effects were effectively antagonized by receptor specific antagonists. It is suggested that 5-HT-induced physiological effects are mediated via distinct classes of receptors that possibly participate in modulation of pigmentary responses of the fish.     

Control of organelle transport in melanophores: regulation of Ca2+ and cAMP levels.

Melanophores of the cichlid Tilapia mossambica can be induced to aggregate pigment by addition of epinephrine to the medium, suggesting adrenergic control of this transport. The melanophore response to adrenergic stimulation was examined using agonists and antagonists that are highly specific for each alpha-adrenoceptor subclass. The signal transduction mechanism of each subclass is unique: stimulation of alpha 1 receptors results in a rise in intracellular free Ca2+, while alpha 2 stimulation results in decreased cAMP levels [Exton, 1985: Am. J. Physiol. 248:E633-E647]. Each alpha 1 or alpha 2 specific agonist tested showed a dose dependent ability to induce aggregation and each was able to effect complete aggregation of pigment, suggesting that aggregation can be mediated either by elevating Ca2+ or by lowering cAMP. However, in the presence of either an alpha 1 or an alpha 2 receptor antagonist, none of the agonists were able to induce significant aggregation, suggesting that changes in levels of both messengers are required for pigment aggregation in the melanophores. Moreover, experiments in which intracellular levels of Ca2+ or cAMP were perturbed, using BAPTA and forskolin, respectively, indicated that elevating Ca2+ in the presence of high cAMP is not sufficient to induce aggregation and, conversely, that lowering cAMP levels in the presence of reduced Ca2+ is not sufficient to induce pigment aggregation. These data indicate that the concentrations of both cAMP and Ca2+ are important in regulating pigment aggregation in teleost melanophores, and suggest that maximal aggregation of pigment requires altering the levels of both messengers.     

Regulation of Norepinephrine Release from Isolated Bovine Irides by Histamine

In the present study, we investigated the effect of histamine on sympathetic neurotransmission from isolated, superfused bovine irides. We also studied the pharmacology of prejunctional histamine receptors that regulate the release of norepinephrine (NE) from this tissue. The effect of exogenous histamine and various histamine receptor agonists was examined on the release of [³H]-norepinephrine ([³H]NE) triggered by electrical field stimulation using the Superfusion Method. Histamine receptor agonists caused a concentration-dependent inhibition of field-stimulated [³H]NE overflow with the following rank order of potency: imetit > histamine > R-α-methylhistamine. In all cases, the inhibitory action of histamine receptor agonists was attenuated at high concentrations of these compounds. The histamine receptor antagonists, clobenpropit (H₃-antagonist/H₄-agonist) and thioperamide (H₃-antagonist) blocked the inhibitory response elicited by R-α-methylhistamine and imetit, respectively. Inhibitory effects of R-α-methylhistamine and clonidine were not additive suggesting that prejunctional H₃- and α₂-adrenoceptors coexist at neurotransmitter release sites. We conclude that histamine produces an inhibitory action on sympathetic neurotransmission in the bovine iris, an effect mimicked by selective H₃-receptor agonists and blocked by H₃-antagonists.     

The effect of pertussis toxin on alpha-2-adrenoceptor-mediated pigment migration in fish melanophores

The aggregation of melanin-granules within fish pigment cells (melanophores) can be elicited either by electrical stimulation of intrinsic nerves or by the addition of adrenergic agonists. The pigment aggregation seems to be mediated by alpha-2-adrenoceptors. In this investigation we have used various agonists and antagonists (noradrenaline, (+)- and (-)-adrenaline, isoprenaline, yohimbine and prazosin) to further characterize the pigment-aggregating receptor of Labrus ossifagus. All the results obtained support the notion of alpha-2-adrenoceptor-mediated pigment aggregation. The pertussis toxin, islet-activating protein (IAP), is known to inhibit the alpha-2-adrenoceptor-mediated signal transduction in mammals. We have used IAP to investigated whether fish melanophore alpha-2-adrenoceptors are also inhibited by this toxin. We found that IAP inactivated the alpha-2-adrenoceptor-mediated pigment aggregation in a dose-dependent manner. The inhibitory IAP-effect had a remarkably short onset-time in the melanophores (maximal effect was obtained within 10 min of incubation). Interestingly, binding of an agonist (noradrenaline) to the receptors prevented IAP from exerting its inhibitory action, whereas binding of an antagonist (yohimbine) gave no protection against the IAP-inactivation. In conclusion, the pigment-aggregating receptors of melanophores of L. ossifagus are very similar to the mammalian alpha-2-adrenoceptors. It is possible to inactivate the melanophore receptor system with IAP and this inactivation has a remarkably short onset-time. Stimulation of the alpha-2-adrenoceptors prevents IAP from inactivating the receptor system.     

Pharmacological characterization of the histamine uptake system in HL-60 human promyelocytic leukemia cells

Serotonin and histamine H₁, H₂ receptor agonists or antagonists inhibited [³H]histamine uptake by HL-60 cells, according to the following order of potency: impromidine >4-MH>histamine>AET>PEA and: cimetidine, histamine>diphenhydramine, serotonin. It is concluded that histamine uptake by HL-60 cells was specifically controlled by the H₂ type histamine receptor and that this active process might be involved in pathophysiological regulations in leukemic and normal granulocytic precursors and in the control of histamine levels in peripheral blood and tissues in man.     

Maxadilan Activates PAC1 Receptors Expressed in Xenopus laevis Melanophores

Functional interactions between ligands and their cognate receptors can be investigated using the ability of melanophores from Xenopus laevis to disperse or aggregate their pigment granules in response to alterations in the intracellular levels of second messengers. We have examined the response of long‐term lines of cultured melanophores from X. laevis to pituitary adenylate cyclase activating peptide (PACAP), a neuropeptide with vasodilatory activity, and maxadilan, a vasodilatory peptide present in the salivary gland extracts of the blood feeding sand fly. Pituitary adenylate cyclase activating peptide increased the intracellular levels of cyclic adenosine monophosphate (cAMP) and induced pigment dispersion in the pigment cells, confirming that melanophores express an endogenous PACAP receptor. Maxadilan did not induce a response in non‐transfected melanophores. When the melanophores were transfected with complementary DNA (cDNA) from the three different members of the PACAP receptor family, maxadilan induced pigment dispersion specifically and cAMP accumulation in melanophores transfected with the cDNA for PAC1 receptors but not VPAC1 or VPAC2 receptors. A melanophore line was generated that stably expresses the PAC1 receptor.     

Subtypes of beta adrenergic receptors mediating pigment dispersion in chromatophores of the medaka, Oryzias latipes

Actions of the adrenergic beta-2 agonists, salbutamol and terbutaline, and the beta-1 antagonists, metoprolol and atenolol, were examined on denervated melanophores and leucophores of a teleost, Oryzias latipes. Beta-2 agonists depressed the pigment-aggregation response of melanophores to norepinephrine, while beta-1 antagonists inhibited the dispersion response of leucophores to isoproterenol but not the melanophore response. These findings suggest that adrenergic receptors mediating pigment dispersion in melanophores are beta-2 and those of leucophores are beta-1. The possible relations between receptor mechanisms and the responses of chromatophores are discussed.     

Up- and down-regulation of membrane receptors as possible mechanisms related to the antiulcer actions of milk in rat gastric mucosa

The effects of a cow's milk diet on receptor activity and histamine metabolism in gastric glands and mucosa isolated from adult rats were examined. The milk diet was associated with (1) a decreased mobilization of H₂ receptors by histamine and (2) an increased mobilization of PGE₂ (prostaglandin E₂) receptors in mucous cells (cytoprotective effect) and parietal cells (antiacid effect). These changes are not observed for the receptors reducing pentagastrin- and histamine-induced gastric acid secretion (pancreatic/enteroglucagons, somatostatin) and stimulating mucus, bicarbonate and pepsin secretions in the rat (secretin). Cimetidine produced a parallel displacement of the histamine dose-response curve, suggesting competitive inhibition between this classical H₂ receptor antagonist and histamine in the two experimental groups. Prostaglandins and other components in milk such as EGF (epidermal growth factor) and somatostatin might therefore protect gastric mucosa by a differential control of PGE₂ and histamine H₂ receptor activity eitherdirectly (PGE₂ in milk) orindirectly (inhibition of endogeneous histamine synthesis/release and stimulation of PGE-I synthesis/release).     

Coexistence of β1- and β2-Adrenoceptors in the Melanophore of the Goby Tridentiger obscurus*

The effects of β-adrenergic agonists and antagonists on the pigmentary state of denervated melanophores in isolated, split, caudal fins of the goby Tridentiger obscurus were examined to investigate the function and the subtype of the β-adrenoceptors of the melanophores. Salbutamol, terbutaline, and dobutamine partially inhibited the pigment-aggregating response of melanophores to norepinephrine. The effects of these β-agonists were inhibited by propranolol. It was confirmed that the melanophores possess both α-and β-adrenoceptors, and that the activation of the β-adrenoceptors induces the dispersion of pigment in the melanophores. Norepinephrine, epinephrine, isoproterenol, dobutamine, salbutamol, and terbutaline evoked the dispersion of pigment in the melanophores in which pigment had previously been aggregated by treatment with verapamil in the presence of phentolamine. The pigment-dispersing effects of two β₁-selective agonists, norepinephrine and dobutamine, were effectively inhibited by metoprolol, a selective antagonist of β₁-receptors. By contrast, the pigment-dispersing effects of two β₂-selective agonists, salbutamol and terbutaline, were not inhibited by metoprolol. Both the effects of nonselective agonists, epinephrine and isoproterenol, were partially inhibited by metoprolol. The actions of all of the β-agonists used were effectively inhibited by propranolol, and they were partially inhibited by butoxamine. These results suggest coexistence of β₁- and β₂-adrenoceptors in the melanophores. The relative numbers of β₁- and β₂-adrenoreceptors as a percentage of the total population of β-adrenoceptors were estimated to be 18.6% and 81.4%, respectively, from analyses of Hofstee plots of the effects of the β-agonists on the melanophores in the presence of butoxamine or metoprolol.     

beta-Adrenergic receptor subtypes in melanophores of the marine gobies Tridentiger trigonocephalus and Chasmichthys gulosus.

The subtype of beta-adrenergic receptors in melanophores of the marine gobies Tridentiger trigonocephalus and Chasmichthys gulosus was studied. Pigment of denervated melanophores in isolated, split caudal fins was preliminarily aggregated by incubating the specimens in a physiological saline containing 10 microM phentolamine and 30-100 microM verapamil or 2-10 nM melatonin, and the responses of the melanophores to a beta-adrenergic agonist added to the incubating medium were recorded photoelectrically. The beta-adrenergic agonists noradrenaline, adrenaline, isoproterenol, salbutamol and, dobutamine were all effective in evoking a dispersion of melanophore pigment in the presence of phentolamine and verapamil or melatonin. The pigment-dispersing effect of noradrenaline (beta 1-selective agonist) was inhibited by metoprolol (beta 1-selective antagonist), propranolol,- and butoxamine. Whereas, the effect of salbutamol (beta 2-selective agonist) was hardly inhibited by metoprolol, though it was considerably inhibited by propranolol and ICI-118551. It was estimated that beta 1- and beta 2-adrenergic receptors coexist at ratios of 8.6:91.4, in the melanophore of Tridentiger trigonocephalus, and 25:75, in the melanophore of Chasmichthys gulosus, through the analyses of Hofstee plots of the effects of the beta-adrenergic drugs. It was suggested that the relation between the pigment-dispersing effect of a beta-adrenergic agonist on the melanophores and the concentration of the drug follows mass action kinetics, when the effect is mainly caused by the activation of beta 2-adrenergic receptors of the melanophores. However, when it is mainly caused by the activation of beta 1-adrenergic receptors of the melanophores, the relation does not follow mass action kinetics.     

Berberine-induced pigment dispersion in Bufo melanostictus melanophores by stimulation of beta-2 adrenergic receptors

Abstract

Reduced production of melanin by decreased or the absence of melanocytes leads to various hypopigmentation disorders, and the development of melanogenetic agents for photoprotection and hypopigmentation disorders is one of the top priority areas of research. Hence, the present study was carried out to elucidate the ability of berberine, a principal active ingredient present in the roots of the herb Berberis vulgaris to stimulate pigment dispersion in the isolated skin melanophores of the toad Bufo melanostictus. In the present study, mean melanophore size index of the isolated skin melanophores of B. melanostictus was assayed after treating with various concentrations of berberine. A marked melanin dispersion response leading to skin darkening was observed in the isolated melanophores of toad in response to berberine, which was found to be mediated through beta-2 adrenergic receptors. The physiologically significant dose-related melanin dispersion effects of berberine per se were found to be completely abolished by propranolol, which is a specific beta-2 adrenergic receptor blocker. These per se melanin dispersal effects were also found to be markedly potentiated by isoprenaline, which is a specific beta-adrenoceptor agonist. The results indicate that berberine causes a tremendous, dose-dependent, physiologically significant pigment dispersing in the isolated skin melanophores of B. melanostictus.     

Biosensing of opioids using frog melanophores

Spectacular color changes of fishes, frogs and other lower vertebrates are due to the motile activities of specialized pigment containing cells. Pigment cells are interesting for biosensing purposes since they provide an easily monitored physiological phenomenon. Melanophores, containing dark brown melanin pigment granules, constitute an important class of chromatophores. Their melanin-filled pigment granules may be stimulated to undergo rapid dispersion throughout the melanophores (cells appear dark), or aggregation to the center of the melanophores (cells appear light). This simple physiological response can easily be measured in a photometer. Selected G protein coupled receptors can be functionally expressed in cultured frog melanophores. Here, we demonstrate the use of recombinant frog melanophores as a biosensor for the detection of opioids. Melanophores were transfected with the human opioid receptor 3 and used for opiate detection. The response to the opioid receptor agonist morphine and a synthetic opioid peptide was analyzed by absorbance readings in an aggregation assay. It was shown that both agonists caused aggregation of pigment granules in the melanophores, and the cells appeared lighter. The pharmacology of the expressed receptors was very similar to its mammalian counterpart, as evidenced by competitive inhibition by increasing concentrations of the opioid receptor inhibitor naloxone. Transfection of melanophores with selected receptors enables the creation of numerous melanophore biosensors, which respond selectively to certain substances. The melanophore biosensor has potential use for measurement of substances in body fluids such as saliva, blood plasma and urine.     

A Rapid Quantitative Bioassay for Evaluating the Effects of Ligands Upon Receptors That Modulate cAMP Levels in a Melanophore Cell Line

A new method for rapidly evaluating the effects of drugs on receptors that regulate intracellular cAMP in a cell line derived from Xenopus laevis melanophores has been developed. Melanophores were plated into sterile 96 well microtiter plates, and 3 days later the cells were treated with melatonin for 30 min to induce melanosome aggregation. Subsequent exposure to MSH or adrenergic agonists caused dose dependent pigment dispersion that peaked within 30 min. The cumulative pigment displacement from cells could be quantitated by using a microplate reader to measure changes in transmittance of light through the wells. The acquired data enabled detailed and reproducible dose response curves and time course analyses to be generated. In addition, the assay followed for the rapid characterization of the effects of antagonists upon the (β adrenergic receptor (β AR). The assay has the potential to test the effects of ligands upon any receptor capable of mediating pigment translocation in the melanophore cell line.     

Activation of carbonic anhydrase isoforms involved in modulation of emotional memory and cognitive disorders with histamine agonists,antagonists and derivatives

Carbonic anhydrases (CAs, EC 4.2.1.1) activators were shown to be involved in memory enhancement and learning in animal models of cognition. Here we investigated the CA activating effects of a large series of histamine based compounds, including histamine receptors (H₁R – H₄R) agonists, antagonists and other derivatives of this autacoid. CA activators may be thus useful for improving cognition as well as in diverse therapeutic areas (phobias, obsessive-compulsive disorder, generalised anxiety, post-traumatic stress disorders), for which activation of this enzyme was recently shown to be involved.     

Multiple α2-Adrenoceptor Signalling Pathways Mediate Pigment Aggregation Within Melanophores

It has previously been shown that α₂-adrenoceptors (α₂-ARs) mediate pigment granule (melanosome) aggregation in melanophores of the teleost fish Labrus ossifagus. The present investigation scrutinized the signalling mechanisms of melanosome aggregation induced by sympathetic nerve stimulation or by exogenous addition of α-AR agonists and cAMP analogues. The following was observed: i) nerve-induced melanosome aggregation was associated with a rapid decrease in the cAMP level; ii) noradrenaline or medetomidine (an α₂-AR agonist) caused melanosome aggregation and reduced the cAMP content; iii) RP-S-CI-cAMP, a membrane-permeating inhibitor of protein kinase A induced melanosome aggregation; and iv) B-HT 920 (an α₂-AR agonist) and methoxamine (an α₁-AR agonist) induced melanosome aggregation, although they did not reduce cAMP. It has been suggested that in some teleost species α₁-ARs mediate melanosome aggregation by increasing the level of intracellular calcium. However, we found that the effect of methoxamine in melanophores from Labrus ossifagus could be blocked by yohimbine (an α₂-AR antagonist) but not by equimolar concentration of prazosin (an α₁-AR antagonist). Furthermore, 1 μM ionomycin (a calcium ionophore) did not induce melanosome aggregation. Our findings therefore do not indicate that α₁-ARs and/or an increase in intracellular calcium mediate melanosome aggregation in Labrus ossifagus. Our results suggest that α₂-AR-mediated melanosome aggregation is induced by multiple signalling pathways. One of these involves a reduction in cAMP, but none involves an increase in intracellular calcium.     

Pharmacological Profiles of the Subtypes of Muscarinic Cholinoceptors That Mediate Aggregation of Pigment in the Melanophores of Two Species of Catfish

Using selective antagonists, including pirenzepine, adiphenine, AF-DX116, gallamine, and 4-DAMP we attempted to characterize the muscarinic cholinoceptors on the melanophores of the translucent glass catfish Kryptopterus bicirrhis and the mailed catfish Corydoras paleatus. The M₃ receptor-selective antagonist, 4-DAMP, potently inhibited the acetylcholine-induced aggregation of pigment in both species. It appeared, therefore, that the receptors that mediated the cholinergically evoked aggregation of melanosomes in these species were of the M₃ muscarinic subtype.     

Calcium Requirement for α-MSH Action on Melanophores: Studies with Forskolin

Abstract

α-MSH-induced pigment dispersion in melanophores shows an absolute requirement for extracellular Ca²⁺. To localize Ca²⁺ sites involved in the mechanism of action of α-MSH we studied the effects of Ca²⁺ deprivation on α-MSH and forskolin-induced melanophore responses. In an in vitro melanophore system employing ventral tailfins of Xenopus tadpoles, melanophore responses were assayed in terms of pigment dispersion and the phosphorylation state of a 53 kDa melanophore-specific protein. In the same melanophore system α-MSH has been shown to specifically increase the phosphorylation of this 53 kDa protein.

Forskolin induces a dose-dependent pigment dispersion (EC₅₀ 7 × 10^?7 M). In contrast to the dispersion induced by α-MSH forskolin-induced dispersion does not require extracellular Ca²⁺. Moreover, in a Ca²⁺-free medium melanophores with permanently activated MSH-receptors aggregate, but can be redispersed by the addition of forskolin. Forskolin increases 53 kDa phosphorylation in a dosedependent manner. Maximal stimulation with forskolin (10^?5 M) is four-fold and equals maximal 53 kDa phosphorylation obtainable with α-MSH. The MSH-induced increase in 53 kDa phosphorylation is inhibited by Ca²⁺ deprivation, whereas the forskolin-induced increase is unaffected. Our results suggest that α-MSH and forskolin stimulate melanophores through a common pathway and confirm that cAMP is a second messenger in α-MSH action in this system. We conclude that the Ca²⁺ sites in the mechanism of α-MSH action on melanophores precede adenylate cyclase activation.     

Role of histamine receptors in the effects of histamine on the production of reactive oxygen species by whole blood phagocytes

Aims

The diverse physiological functions of histamine are mediated through distinct histamine receptors. In this study we investigated the role of H₂R and H₄R in the effects of histamine on the production of reactive oxygen species by phagocytes in whole blood.

Main methods

Changes in reactive oxygen species (ROS) production by whole blood phagocytes after treatment with histamine, H₄R agonists (4-methylhistamine, VUF8430), H₂R agonist (dimaprit) and their combinations with H₄R antagonist (JNJ10191584) and H₂R antagonist (ranitidine) were determined using the chemiluminescence (CL) assay. To exclude the direct scavenging effects of the studied compounds on the CL response, the antioxidant properties of all compounds were measured using several methods (TRAP, ORAC, and luminol–HRP–H₂O₂ based CL).

Key findings

Histamine, 4-methylhistamine, VUF8430 and dimaprit inhibited the spontaneous and OZP-activated whole blood CL in a dose-dependent manner. On the other hand, only VUF8430 was able to inhibit PMA-activated whole blood CL. Ranitidine, but not JNJ10191584, completely reduced the effects of histamine, 4-methylhistamine and dimaprit. The direct scavenging ability of tested compounds was negligible.

Significance

Our results demonstrate that the inhibitory effects of histamine on ROS production in whole blood phagocytes were caused by H₂R. Our results also suggest that H₄R agonists in concentrations higher than 10^− 6 M may also influence ROS production via binding to H₂R.