Usefulness of testing histamine release from isolated human basophils in anti-allergic and anti-asthmatic drug assessment]

Histamine release from isolated human basophils test was used to evaluate an activity of: histamine receptors H1 and H2 blockers, agonists of beta-receptors, calcium channel blocking agents, hydrocortisone, and disodium cromoglycate (Intal). The study involved 84 patients hospitalized for the bronchial asthma. Basophils were isolated with Day's technique modified by Shov and Norn. Histamine was measured with Shov's spectrofluorimetric technique. It was found that histamine release from isolated human basophils may be used in both evaluation of the mechanism of action and efficiency of drugs used in allergic diseases therapy.     

Very low density lipoproteins (VLDL) trigger the release of histamine from human basophils

Circulating basophils are well established sources of the granule-associated mediator, histamine. The physiological control, however, of histamine release from human basophils is poorly understood. Because histamine may play a role in the transendothelial transport of various compounds, including very low density lipoprotein (VLDL) and its hydrolysis products, we investigated the possibility that VLDL regulates mediator release from basophils. The incubation of VLDL (at physiological concentrations) with basophils (isolated as mixed leukocyte preparations) resulted in a significant release of histamine. Histamine release was dependent on VLDL concentration (half-maximal stimulation occurring at VLDL-protein concentration of 15-20 micrograms/ml), length of incubation (half-maximal release at 5-12 min), temperature (37 degrees C optimum) and required calcium (concentration 0.5-2.0 mM). Furthermore, VLDL-induced histamine release was inhibited by three different mediator-release inhibitors: dimaprit, dibutyryl cAMP and nordihydroguaiaretic acid. Incubation of basophils with LDL or HDL under the same experimental conditions did not result in significant histamine release from basophils. The histamine-secretory response of basophils obtained from different donors varied considerably. Basophils isolated from 28 donors and challenged with 100 micrograms/ml VLDL released 23 +/- 5% of their cellular histamine (mean +/- S.E.; with a range of 0-94%). Desensitization of VLDL-induced histamine release could be accomplished by preincubation of basophils with either VLDL or anti-IgE but not with N-formyl-L-methionyl-L-leucyl-L-phenylalanine. Through the secretion of histamine, a potent vasoactive mediator (and also possibly through granule-associated glycosaminoglycans, stimulants of the enzyme lipoprotein lipase), this novel effect of VLDL may be part of a physiological loop for the regulation of VLDL hydrolysis and lipid transport. This effect of VLDL may also have deleterious consequences, because of the atherogenic properties of histamine.     

Effect of calcium channel blockers on histamine liberation from isolated basophils and bronchial reactivity in patients with bronchial asthma]

An effect of verapamil and nifedipine on the cellular histamine release and non-specific reactivity of the bronchi measured with methacholine test was studied. In vitro tests were carried out in basophils isolated from 19 asthmatic patients. Reactivity of the bronchi was investigated in 5 patients. Both verapamil and nifedipine significantly inhibited histamine release from the cells in vitro in all examined concentrations (10(-7)-10(-4) M) while a single dose of these drugs administered to the asthmatic patients did not exert a significant effect on the airway constriction in vivo.     

IL-8 inhibits histamine release from human basophils induced by histamine-releasing factors, connective tissue activating peptide III, and IL-3.

We have previously purified and partially characterized histamine releasing factors (HRF), which were derived from a mixture of human mononuclear cells and platelets. We now report the effect of IL-8 upon HRF-, connective tissue activating peptide III (CTAP III)-, and IL-3-induced histamine release from human basophils. We determined that IL-8 itself, at concentrations between 10(-7) to 10(-11) M, does not release histamine from basophils, although positive results are observed in two of 26 subjects at 10(-7) M. Unfractionated (crude) HRF released histamine in 25 of 26 donors, in the range of 6.7% to 100% of total basophil histamine stores. When basophils were preincubated with IL-8 (10(-7) to 10(-11) M) for 5 min, followed by a 40-min incubation with HRF, histamine release was significantly inhibited in 20 of 25 donors. Inhibition was observed at as little as 10(-11) M IL-8, with maximal inhibition being attained at 10(-9) M. HRF-containing supernatants contain a mixture of different histamine-releasing moieties. To better define which factor(s) may be inhibited by IL-8, fractionated supernatants, purified CTAP III, and IL-3 were studied. Histamine release produced by two different HRF-containing chromatographic fractions (HRFvoid and HRFpeak 2) and purified CTAP-III (5 micrograms/ml) was inhibited by IL-8 in 10 of 12 donors, three of three donors, and seven of 10 donors, respectively. IL-3 (5000 U/ml)-dependent histamine release was inhibited by IL-8 in all subjects tested. In contrast, histamine release by anti-IgE and FMLP was not affected by IL-8. Thus, IL-8 appears to be an inhibitor of cytokine-like molecules that induce histamine release and may represent the previously described 8-kDa histamine release inhibitory factor present in mononuclear cell supernatants.     

Histamine release by chemotactic, formyl methionine-containing peptides.

Certain formyl dipeptides and tripeptides containing methionine released histamine from human basophils at concentrations of 10(-4) to 10(-7) M. However, N-formyl amino acids did not release histamine. Tripeptides, in general, were more active than dipeptides. An acyl group was required for histamine release although an N-terminal position for Met was not essential. Histamine release from human basophils by these peptides correlated well with their chemotactic activity for rabbit leukocytes.     

Possible role of calmodulin in the control of histamine release from human basophil leukocytes

We investigated the possible role of calmodulin (CaM) in the control of histamine release from human basophil leukocytes using several CaM antagonists. Trifluoperazine (TFP) (10(-6)-2 X 10(-5) M), pimozide (10(-6)-1.5 X 10(-5) M), chlorpromazine (CPZ) (10(-5)-10(-4) M) and promethazine (PMZ) (2 X 10(-5)-10(-4) M) inhibited in vitro histamine secretion from human basophils induced by several immunological (antigen, anti-IgE, and formyl-L-methionyl-L-leucyl-L-phenylalanine: f-met peptide) and nonimmunological (Ca2+ ionophore A23187 and the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate: TPA) stimuli. Trifluoperazine sulfoxide (TFP-S) and chlorpromazine sulfoxide (CPZ-S), which have very low affinity to CaM, had practically no inhibitory effect on histamine release from human basophils. The inhibitory effect of TFP could be made irreversible by irradiating the cells with UV light. A sulfonamide derivative, the compound N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) (2.5 X 10(-5)-2 X 10(-4) M), which selectively binds to CaM, inhibited the release of histamine from basophils. In contrast, the chloride deficient analogue, W-5, which interacts only weakly with CaM, had practically no inhibiting effect. The IC50 for enzyme release by a series of eight CaM antagonists was closely correlated (r = 0.91; p less than 0.001) with the CaM specific binding, supporting the concept that these agents act by binding to CaM and thereby inhibiting histamine release. TFP and W-7 inhibited histamine release in the absence and in the presence of increasing concentrations of extracellular Ca2+. These results emphasize the possible role of CaM in the control of histamine secretion from human basophils.     

Morphologic and immunologic characterization of human basophils developed in cultures of cord blood mononuclear cells

Selective growth of human basophilic granulocytes was obtained in suspension cultures of mononuclear cells from umbilical cord blood. Approximately 50 to 80% of nonadherent cells recovered from 2- to 3-wk-old cultures contained metachromatic granules, and these cells were identified as human basophilic granulocytes by electron microscopy. Histamine content of cultured human basophils was comparable to that in peripheral blood basophils. Cultured basophils bear 2.7 to 3.7 X 10(5) IgE receptors per cell that bind both human IgE and rodent IgE with comparable affinity. Average equilibrium constants of the receptors for human IgE and mouse IgE were 2.56 +/- 0.88 X 10(9) M-1 and 1.85 +/- 0.86 X 10(9) M-1, respectively. The cell-surface component of the IgE receptors on cultured basophils has a m.w. of 64,000. Cultured basophils could be passively sensitized with human IgE and mouse IgE monoclonal antibody, and sensitized basophils released characteristic cytoplasmic granules and both histamine and arachidonate upon challenge with either anti-human IgE or antigen. Incubation of cultured basophils with ionophore A23187 or F-Met-Leu-Phe resulted in histamine release. However, compound 48/80 failed to induce histamine release from the cells.     

FK-506, a potent novel inhibitor of the release of proinflammatory mediators from human Fc epsilon RI+ cells.

FK-506, a macrolide that binds with high affinity to a specific binding protein, and the structurally related macrolide rapamycin (RAP) were compared to cyclosporin A (CsA) for their effects on the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4) inflammatory mediators from human basophils. FK-506 (1 to 300 nM) concentration dependently inhibited histamine release from basophils activated by Der p I Ag, anti-IgE, or compound A23187. FK-506 was more potent than CsA when basophils were challenged with Ag (IC50 = 25.5 +/- 9.5 vs 834.3 +/- 79.8 nM; p less than 0.001), anti-IgE (IC50 = 9.4 +/- 1.7 vs 441.3 +/- 106.7 nM; p less than 0.001), and A23187 (IC50 = 4.1 +/- 0.9 vs 36.7 +/- 3.8 nM; p less than 0.001). The maximal inhibitory effect of FK-506 was higher than that caused by CsA when basophils were activated by Der p I (80.0 +/- 3.6 vs 49.5 +/- 4.7%; p less than 0.001) and anti-IgE (90.4 +/- 1.8 vs 62.3 +/- 2.9%; p less than 0.001). FK-506 had little or no effect on the release of histamine caused by f-met peptide, phorbol myristate (12-tetradecanoyloxy-13-acetoxy-phorbol), and bryostatin 1. RAP (30 to 1000 nM) selectively inhibited only IgE-mediated histamine release from basophils, although it had no effect on mediator release caused by f-met peptide, A23187, 12-tetradecanoyloxy-13-acetoxy-phorbol, and bryostatin 1. FK-506 also inhibited the de novo synthesis of sulfidopeptide leukotriene C4 from basophils challenged with anti-IgE. Low concentrations of FK-506 and CsA synergistically inhibited the release of mediators from basophils induced by anti-IgE or compound A23187. IL-3 (3 and 10 ng/ml), but not IL-1 beta (10 and 100 ng/ml), reversed the inhibitory effect of both FK-506 and CsA on basophils challenged with anti-IgE or A23187. RAP was a competitive antagonist of the inhibitory effect of FK-506 on A23187-induced histamine release from basophils with a dissociation constant of about 30 nM. In contrast, RAP did not modify the inhibitory effect of CsA on A23187-induced histamine release. These data indicate that FK-506 is a potent antiinflammatory agent that acts on human basophils presumably by binding to a receptor site (i.e., FK-506 binding protein).     

Protein L. A bacterial Ig-binding protein that activates human basophils and mast cells.

Peptostreptococcus magnus strain 312 (10(6) to 10(8)/ml), which synthesizes a protein capable of binding to kappa L chains of human Ig (protein L), stimulated the release of histamine from human basophils in vitro. P. magnus strain 644, which does not synthesize protein L, did not induce histamine secretion. Soluble protein L (3 x 10(-2) to 3 micrograms/ml) induced histamine release from human basophils. The characteristics of the release reaction were similar to those of rabbit IgG anti-Fc fragment of human IgE (anti-IgE): it was Ca2(+)- and temperature-dependent, optimal release occurring at 37 degrees C in the presence of 1.0 mM extracellular Ca2+. There was an excellent correlation (r = 0.82; p less than 0.001) between the maximal percent histamine release induced by protein L and that induced by anti-IgE, as well as between protein L and protein A from Staphylococcus aureus (r = 0.52; p less than 0.01). Preincubation of basophils with either protein L or anti-IgE resulted in complete cross-desensitization to a subsequent challenge with the heterologous stimulus. IgE purified from myeloma patients PS and PP (lambda-chains) blocked anti-IgE-induced histamine release but failed to block the histamine releasing activity of protein L. In contrast, IgE purified from myeloma patient ADZ (kappa-chains) blocked both anti-IgE- and protein L-induced releases, whereas human polyclonal IgG selectively blocked protein L-induced secretion. Protein L acted as a complete secretagogue, i.e., it activated basophils to release sulfidopeptide leukotriene C4 as well as histamine. Protein L (10(-1) to 3 micrograms/ml) also induced the release of preformed (histamine) and de novo synthesized mediators (leukotriene C4 and/or PGD2) from mast cells isolated from lung parenchyma and skin tissues. Intradermal injections of protein L (0.01 to 10 micrograms/ml) in nonallergic subjects caused a dose-dependent wheal-and-flare reaction. Protein L activates human basophils and mast cells in vitro and in vivo presumably by interacting with kappa L chains of the IgE isotype.     

Cyclosporin A rapidly inhibits mediator release from human basophils presumably by interacting with cyclophilin.

We have examined the effects of cyclosporin A (CsA) and a series of CsA analogs that bind with decreasing affinity to cyclophilin, to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4; LTC4) mediators of inflammatory reactions from human basophils. CsA (8 to 800 nM) concentration-dependently inhibited (5 to 60%) histamine release from peripheral blood basophils challenged with anti-IgE. CsA was more potent (92.6 +/- 1.8 vs 59.1 +/- 4.5%; p less than 0.001) and, at low concentrations, more effective when the channel-operated influx of Ca2+ was bypassed by the ionophore A23187 (IC40 = 24.1 +/- 3.9 vs 105.5 +/- 22.2 nM; p less than 0.05). CsA had no effect on the release of histamine caused by phorbol myristate and bryostatin 1 that activate different isoforms of protein kinase C. Inhibition of histamine release from basophils challenged with anti-IgE was not abolished by washing (three times) the cells before anti-IgE challenge. CsA also inhibited the de novo synthesis of LTC4 from basophils challenged with anti-IgE. The inhibitory effect of CsA was very rapid, and the drug, added from 1 to 10 min during the reaction, inhibited the ongoing release of histamine caused by anti-IgE and by A23187. The experiments with CsA analogs (CsG, CsC, CsD, and CsH) showed that CsH, which has an extremely low affinity for cyclophilin, has no effect on basophil mediator release. In addition, there is a significant correlation between the concentrations of CsA, G, C, and D that inhibited by 30% the histamine release induced by anti-IgE (r = 0.99; p less than 0.001) and by A23187 (r = 0.87; p less than 0.001) and their affinity for cyclophilin.     

Human basophil/mast cell releasability. VII. Heterogeneity of the effect of adenosine on mediator secretion

5'-N-ethylcarboxamideadenosine (NECA) greater than 2-chloroadenosine greater than adenosine greater than N6-(R-phenyl-isopropyl)-adenosine (R-PIA) inhibited in vitro anti-IgE-induced histamine and peptide leukotriene C4 (LTC4) release from human basophils in a concentration-dependent fashion. Micromolar concentrations of adenosine, NECA and R-PIA potentiated the anti-IgE-stimulated release of histamine and LTC4 from human lung parenchymal mast cells. Submillimolar concentrations of adenosine, NECA and R-PIA inhibited in a concentration dependent manner the release of histamine and prostaglandin D2 (PGD2) from skin mast cells challenged with anti-IgE. These results demonstrate marked heterogeneity of the modulatory effect exerted by adenosine on mediator release from human basophils and mast cells.     

Induction of histamine secretion by polycations

Poly(arginine), poly(lysine) and poly(ornithine) induce histamine secretion from human basophil leukocytes in the concentration range 1--100 nmol/l. Histamine secretion induced by poly(arginine) requires extracellular calcium at 0.1--1 mmol/l. Strontium (1--10 mmol/l) will substitute for calcium. Lanthanum (30--90 nmol/l) inhibits histamine release induced by poly(arginine). Histamine secretion induced by poly(arginine) is inhibited by 1--30 mumol/l N-ethyl-maleimide, 0.3--3 mmol/l 2-deoxy-D-glucose, 0.3--3 mmol/l dibutyryl cyclic AMP, 0.3--3 mmol/l, adenosine 3'5'-cyclicphosphorothioate. The action of poly(arginine) is inhibited by pretreatment of basophils at 47 degrees C or with neuraminidase. 10 microgram/ml heparin inhibits the response to poly(arginine). Histamine releasing potency of the polymer amino acids is dependent on chain length of the peptide. Succinylated poly(lysine) is inactive. Monomer amino acids do not release histamine and do not inhibit the action of the polymers. Histones and protamine do not release histamine, nor do the peptides eledoisin and tuftsin. Putrescine, cadaverine, spermine and spermidine do not release histamine. Poly(glutamic acid), poly(aspartic acid) and poly(tyrosine) are also inactive. The IgE-mediated release of histamine appears to be independent of that mediated by poly(arginine).     

Anti-human IgG causes basophil histamine release by acting on IgG-IgE complexes bound to IgE receptors.

We have reexamined the ability of anti-human IgG antibodies to induce histamine release from human basophils. A panel of purified murine mAbs with International Union of Immunological Societies-documented specificity for each of the four subclasses of human IgG was used. Of the 24 allergic subjects studied, the basophils of 75% (18/24) released greater than 10% histamine to one or more anti-IgG1-4 mAb, whereas none of the 13 nonatopic donor's basophils released histamine after stimulation with optimal amounts of anti-IgG mAb. The basophils of 85% (11/13) of the nonatopic donors did respond to anti-IgE challenge, as did 92% (22/24) of the atopic donor cells. Histamine release was induced most frequently by anti-IgG3, and 10/18 anti-IgG responder cells released histamine with mAb specific for two or more different subclass specificities. The rank order for induction of histamine release was anti-IgG3 greater than anti-IgG2 greater than IgG1 greater than anti-IgG4. As in our previous study using polyclonal anti-IgG, 100- to 300-micrograms/ml quantities of the anti-IgG mAb were required for maximal histamine release, about 1000-fold higher than those for comparable release with anti-human IgE. Specificity studies using both immunoassays and inhibition studies with IgE myeloma protein indicated that anti-IgG induced histamine release was not caused by cross-reactivity with IgE. Ig receptors were opened by lactic acid treatment so that the cells could be passively sensitized. Neither IgE myeloma nor IgG myeloma (up to 15 mg/ml) proteins could restore the response to anti-IgG mAb. However, sera from individuals with leukocytes that released histamine upon challenge with anti-IgG mAb could passively sensitize acid-treated leukocytes from both anti-IgG responder and nonresponder donors for an anti-IgG response. The only anti-IgG mAb that induced release from these passively sensitized cells were those to which the serum donor was responsive. Sera from non-IgG responders could not restore an anti-IgG response. These data led to the hypothesis that the IgG specific mAb were binding to IgG-IgE complexes that were attached to the basophil through IgE bound to the IgE receptor. This was shown to be correct because passive sensitization to anti-IgG could be blocked by previous exposure of the basophils to IgE. We conclude that anti-IgG-induced release occurs as a result of binding to IgG anti-IgE antibodies and cross-linking of the IgE receptors on basophils.     

Characterization of the anti-inflammatory effect of FK-506 on human mast cells.

We have examined the effects of FK-506 and of the struturally related macrolide rapamycin, which bind with high affinity to a specific binding protein (FKBP), to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized inflammatory mediators (sulfidopeptide leukotriene C4 and prostaglandin D2) from mast cells isolated from human lung parenchyma. FK-506 (0.1 to 300 nM) concentration dependently inhibited histamine release from lung parenchymal mast cells activated by anti-IgE. FK-506 was more potent in lung mast cells than in basophils (IC50 = 1.13 +/- 0.46 nM vs 5.28 +/- 0.88 nM; p less than 0.001), whereas the maximal inhibitory effect was higher in basophils than in lung mast cells (88.4 +/- 2.5% vs 76.4 +/- 3.8%; p less than 0.01). FK-506 had little or no inhibitory effect on histamine release from lung mast cells challenged with compound A23187, whereas it completely suppressed A23187-induced histamine release from basophils. FK-506 also inhibited the de novo synthesis of 5-lipoxygenase (sulfidopeptide leukotriene C4) and cyclo-oxygenase (prostaglandin D2) metabolites of arachidonic acid from mast cells challenged with anti-IgE. Unlike in basophils, Il-3 (3 to 30 ng/ml) did not modify anti-IgE- or A23187-induced histamine release from lung mast cells nor did it reverse the inhibitory effect of FK-506. Rapamycin (3 to 300 nM) had little or no effect on the release of histamine from lung mast cells, but it was a competitive antagonist of the inhibitory effect of FK-506 on anti-IgE-induced histamine release from human mast cells with a dissociation constant of about 12 nM. These data indicate that FK-506 is a potent anti-inflammatory agent that acts on human lung mast cells presumably by binding to a receptor site (i.e., FKBP).     

Leukotriene C4 formation by enriched human basophil preparations from normal and asthmatic subjects.

Numbers of circulating basophils are increased in asthmatic subjects, compared to normal subjects. Basophil enriched cell preparations from normal and asthmatic subjects were challenged in vitro with the calcium ionophore A23187, anti-IgE, or opsonized zymosan to study leukotriene C4 formation, histamine release, and prostaglandin D2 formation. No prostaglandin D2 formation by basophils was observed. Furthermore, opsonized zymosan was not capable of inducing any mediator formation or release from basophils. At optimal stimulation conditions no differences were found between basophils from normal and asthmatic subjects concerning A23187 or anti-IgE induced leukotriene C4 formation or histamine release. A23187 and anti-IgE induced leukotriene C4 formation were in the range of 1-20 and 0.6-4.8 pmol/10(6) basophils respectively.     

Histamine releasibility and expression of Lyn and Syk kinases in Indian subjects and role of less potent IL-3 in non-releaser basophils

BACKGROUND: Allergen-mediated activation of the IgE signal pathway in basophils and mast cells leads to release of mediators in-vitro and in-vivo systems. However, basophils from 10% to 20% of the population do not release histamine and other mediators on activation of the IgE signal transduction pathway and this has been attributed to the absence of tyrosine kinases Lyn and Syk. Interestingly, when these non-releaser basophils are incubated with the IL-3, it leads to the recovery of the histamine releasibility. OBJECTIVE: To investigate histamine releasibility in the Indian population and to evaluate the role of IL-3 with reference to non-releaser phenotypes. METHODS: Peripheral blood basophils from healthy adults were purified by density gradient centrifugation and negative immuno-selection. Histamine release assay was performed fluorometrically. Assessment of Lyn and Syk expression were carried out by flow-cytometry. SNP analysis in the IL-3 gene was carried by sequencing analysis. RESULTS: Histamine release after ConA challenge varied greatly from 0% to 100% in Indian subjects. Eighteen percent subjects showed less than 5% histamine release (non-releasers). Flow-cytometric analysis revealed a significantly reduced expression of Lyn and Syk kinases in basophils (p<0.05). Histamine release also significantly correlated with expression of Lyn and Syk kinase (p<0.05). Non-releasers showed the presence of SNP at +79 (T-C), which leads to the one amino acid change at 8th position in the mature IL-3 from serine to proline. CONCLUSIONS: About 18% of the Indian subjects studied showed non-releaser phenotype and also had reduced Lyn and Syk kinase expression. Non-releasers have also shown the presence of less potent isoform of IL-3/P8, which is suspected to be responsible for the non-releaser phenotype. This needs to be extended to a larger sample size and could be a potential target for the development of therapeutics for allergic patients.     

Human monocytes generate basophil histamine-releasing activities

Human peripheral blood monocytes generated activities during 24-h culture that were capable of triggering histamine release from 17 of 18 human basophil donors. Monocytes and their in vitro transformed macrophages continued to elaborate these basophil histamine-releasing activities for at least 3 wk in culture. In the 18 basophil donors tested, maximum histamine release induced by monocyte supernatants was 33.8 +/- 5.9% (mean +/- SEM) of total basophil histamine content; optimum anti-IgE-induced release was 38.8 +/- 6.2%. Basophil histamine release in response to monocyte activities was optimal at 37 degrees C and at calcium concentrations of 2 to 5 mM. Release was greater than 90% complete 1 min after challenge and was inhibited by anti-allergic drugs. The mechanism of release appeared to be independent of IgE binding. Gel filtration of supernatants derived from both day 1 (monocyte stage) and day 14 (macrophage stage) cultures demonstrated activity peaks with approximate m.w. of 12,000 and 30,000. In contrast to the marked responsiveness of basophils, only 2 of 10 human lung mast cell preparations responded; release in those preparations was low: 3% and 13% histamine release, respectively. Thus, monocytes produce potent histamine-releasing activities with differential actions on basophils and mast cells.     

Ontogeny of the smooth muscle response to histamine in rabbit and human small bowel

Histamine is known to stimulate small bowel smooth muscle contraction in adults. We studied the response to histamine of small bowel from 27 day gestation fetal (term = 31 days), 4 days-old neonatal, and weanling rabbits using an isolated muscle strip technique in vitro. Sensitivity to histamine stimulation decreased with increasing age, with a six-fold difference in mean D50 between fetal and weanling bowel. A strong contractile response was also obtained in human fetal bowel between 16 and 24 weeks gestation. The response to histamine was inhibited at all ages in both rabbit and human bowel by specific histamine H1 receptor blockade with diphenhydramine, and not by H2 or cholinergic receptor blockers. We conclude that histamine stimulates rabbit and human fetal small bowel contraction; stimulation specifically occurs via the histamine H1 receptor, and is unrelated to release of acetylcholine; sensitivity to histamine decreases significantly with maturity in the rabbit.     

Histamine-stimulated and GTP-binding proteins-mediated phospholipase A2 activation in rabbit platelets

Histamine is known to be a mediator of inflammation. In order to understand the role of histamine in platelets, we have examined the effects of histamine on arachidonic acid (AA) release, cAMP accumulation, inositol trisphosphate production, and serotonin secretion. Incubation of rabbit (and human) platelets with histamine resulted in rapid increase of [3H]AA release from the platelets prelabeled with [3H]AA. The effect of histamine was blocked by the addition of H1 receptor antagonist mepyramine. Histamine did not substantially affect the cAMP content and inositol trisphosphate production. Histamine-stimulated AA release was not observed in digitonin-permeabilized platelets, whereas histamine acted synergistically with GTP or GTP analog, guanosine 5'-(3-O-thio)triphosphate. Histamine-stimulated, and GTP analog-dependent AA release was inhibited by guanosine 5'-(2-O-thio) diphosphate. The effects of three receptor stimulants, thrombin, norepinephrine, and histamine were both diminished by 1 microgram/ml of pertussis toxin treatment and by the antiserum against GTP-binding proteins (G proteins) treatment. However, the antiserum against beta gamma subunits of G proteins inhibited the histamine effect, not thrombin effect. 4 beta-Phorbol 12-myristate 13-acetate (PMA) treatment enhanced histamine-stimulated AA release and serotonin secretion but inhibited thrombin-stimulated reactions. The effect of PMA was dose dependent and was due to enhance the coupling of histamine receptors and G proteins. The results show the existence of H1 histamine receptors which couple phospholipase A2 activation via pertussis toxin-sensitive G proteins. Histamine actions differ in sensitivities to anti-beta gamma antiserum treatment and PMA treatment from thrombin actions.