Histamine Pretreatment Research Articles

Subthreshold Doses of Inflammatory Mediators potentiate one another to Elicit Reflex Cardiorespiratory Responses in Anesthetized Rats.

Reflex cardio-vascular and respiratory (CVR) alterations evoked by intra-arterial instillation of nociceptive agents are termed vasosensory reflexes. Such responses elicited by optimal doses of inflammatory mediators have been described in our earlier work. The present study was designed to evaluate the interactions between subthreshold doses of inflammatory mediators on perivascular nociceptive afferents in urethane anesthetized rats. Healthy male adult rats (Charles-Foster strain) were anesthetized with an intraperitoneal injection of urethane. After anesthesia, the right femoral artery was cannulated. Respiratory movements, blood pressure, and electrocardiogram were recorded. The interactions between subthreshold doses of algogens in the elicitation of vasosensory reflex responses were studied by instillation of bradykinin (1 nM) and histamine (100 µM) into the femoral artery one after the other, in either temporal combination in separate groups of rats. The CVR responses obtained in these groups were then compared with the responses produced by 100 µM histamine and 1 nM bradykinin in saline-pretreated groups, which served as control. Subthreshold doses of histamine elicited transient tachypnoeic, hyperventilatory, hypotensive, and bradycardiac responses, in rats pretreated with subthreshold doses of bradykinin [P <0.01, two-sided Dunnett's test] but not in saline pretreated groups [P >0.05, two-sided Dunnett's test]. Similar responses were elicited by bradykinin after histamine pretreatment compared to the saline-pretreated group. Furthermore, CVR responses produced by histamine in the bradykinin-pretreated group were greater in magnitude as compared to bradykinin-induced responses in the histamine-pretreated group [P <0.05, two-sided Dunnett's test]. The present study demonstrates that both bradykinin and histamine potentiate one another in the elicitation of vasosensory reflex responses, and bradykinin is a better potentiator than histamine at the level of perivascular nociceptive afferents in producing reflex CVR changes.

Histidine Decarboxylase Deficiency Causes Tourette Syndrome: Parallel Findings in Humans and Mice

Tourette syndrome (TS) is characterized by tics, sensorimotor gating deficiencies, and abnormalities of cortico-basal ganglia circuits. A mutation in histidine decarboxylase (Hdc), the key enzyme for the biosynthesis of histamine (HA), has been implicated as a rare genetic cause. Hdc knockout mice exhibited potentiated tic-like stereotypies, recapitulating core phenomenology of TS; these were mitigated by the dopamine (DA) D2 antagonist haloperidol, a proven pharmacotherapy, and by HA infusion into the brain. Prepulse inhibition was impaired in both mice and humans carrying Hdc mutations. HA infusion reduced striatal DA levels; in Hdc knockout mice, striatal DA was increased and the DA-regulated immediate early gene Fos was upregulated. DA D2/D3 receptor binding was altered both in mice and in humans carrying the Hdc mutation. These data confirm histidine decarboxylase deficiency as a rare cause of TS and identify HA-DA interactions in the basal ganglia as an important locus of pathology.

Adaptive Changes in the Heterologous Desensitization Event after Histamine Activates its H1 Receptor in C6 Glioma Cells

The possible role of protein kinase C (PKC) linked to the heterologous desensitization of histamine H1 and other receptors was investigated. Receptor activators, such as ATP, PAF, and bradykinin, or calcium ionophore A-23187 were employed for study in the cultured C6 glioma cells. Cells were loaded with the calcium-sensitive fluorescent dye fura-2 and increases in intracellular free calcium concentration ([Ca^(2+)](subscript i)) were monitored in response to histamine H1 or other receptor activations. Histamine stimulated the release of Ca^(2+) from intracellular Ca^(2+) stores and Ca^(2+) influx across the plasma membrane. Under pre-activation of PKC with the phorbol ester β-phorbol-12, 13 dibutyrate (PMA, 10 nM), it caused an attenuation of histamine (100 μM), ATP (100 μM), PAF (1 μM), or bradykinin (1 μM)-induced [Ca^(2+)](subscript i) increase. The relative potency order of attenuation was histamine (92.2%) ＞ ATP (83.3%) ＞ PAF (50.2%) ＞ bradykinin (48.0%) ＞ A23187 (0.2%) in the calcium movement. Similar trend was observed in the histamine pretreatment case among these agents. In this study, 100 nM A23187 (its action is independent of protein kinase C activity), was not found to have a heterologous desensitization in histamine- or PMA-induced calcium movement. Hence PKC activator PMA, and its inhibitor staurosporine could cause significant effects on this heterologous desensitization event. It is proposed that PKC phosphorylating the substrate proteins plays an important role in this shortterm desensitization of histamine acting on histamine H1 receptors in the C6 glioma cells. Our data support the claim that PKC phosphorylates its substrate of the inositol 1, 4, 5-trisphosphate receptor (IP3R) and decreases the mobilization of [Ca^(2+)](subscript i) in these cells, which linked to homologous or/and heterologous desensitization.

The Affinity of Histamine for Gq Protein-Coupled Histamine H1-Receptors Is Predominantly Regulated by Their Internalization in Human Astrocytoma Cells

We examined the regulatory mechanisms of the affinity of Gq protein-coupled histamine H1-receptors for histamine after histamine pretreatment in intact human U373 MG astrocytoma cells. In control cells, the displacement curves for histamine against the binding of 5 nM [3H] mepyramine, a radioligand for H1-receptors, showed the presence of two binding sites for histamine, that is, high and low affinity sites. Pretreatment with 0.1 mM histamine for 30 min at 37°C induced a significant reduction in the percentage of high affinity sites for histamine and a concomitant increase in the percentage of low affinity sites with no change in their pIC50 values. These histamine-induced changes were insensitive to 30 μM KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase II, but they were completely inhibited either by 0.4 mM ZnCl2, an inhibitor of G protein-coupled receptor kinases (GRKs), or under hypertonic conditions, where clathrin-mediated endocytosis is known to be inhibited. These results suggest that histamine-induced conversion of high to low affinity sites for histamine is predominantly regulated by GRK/clathrin-mediated internalization of H1-receptors in human astrocytoma cells.

Histamine H1 Receptor Induces Cytosolic Calcium Increase and Aquaporin Translocation in Human Salivary Gland Cells

One of the common side effects of antihistamine medicines is xerostomia (dry mouth). The current consensus is that antihistamine-induced xerostomia comes from an antimuscarinic effect. Although the effect of antihistamines on salivary secretion is both obvious and significant, the cellular mechanism whereby this happens is still unclear because of the lack of knowledge of histamine signaling in human salivary glands. Here, we have studied histamine receptors and the effect of antihistamines on human submandibular acinar cells. In primary cultured human submandibular gland and a HSG cell line, histamine increased the intracellular Ca(2+) concentration. The histamine-induced cytosolic free Ca(2+) concentration ([Ca(2+)](i)) increase was inhibited by histamine H1 receptor-specific antagonists, and the expression of the functional histamine H1 receptor was confirmed by reverse transcription-polymerase chain reaction. Interestingly, histamine pretreatment did not inhibit a subsequent carbachol-induced [Ca(2+)](i) rise without "heterologous desensitization." Chlorpheniramine inhibited a carbachol-induced [Ca(2+)](i) increase at a 100-fold greater concentration than histamine receptor antagonism, whereas astemizole and cetrizine showed more than 1000-fold difference, which in part explains the xerostomia-inducing potency among the antihistamines. Notably, histamine resulted in translocation of aquaporin-5 to the plasma membrane in human submandibular gland cells and green fluorescent protein-tagged aquaporin-5 expressing HSG cells. We found that histidine decarboxylase and the histamine H1 receptor are broadly distributed in submandibular gland cells, whereas choline acetyltransferase is localized only at the parasympathetic terminals. Our results suggest that human salivary gland cells express histamine H1 receptors and histamine-synthesizing enzymes, revealing the cellular mechanism of antihistamine-induced xerostomia.

Intact Cell Binding for In Vitro Prediction of Sedative and Non-sedative Histamine H1–Receptor Antagonists Based on Receptor Internalization

We evaluated changes in the binding properties of sedative and non-sedative histamine H1–receptor antagonists induced by internalization of H1 receptors in intact human U373 MG astrocytoma cells. Internalization of H1 receptors was induced without their degradation by treatment with 0.1 mM histamine for 30 min at 37°C, and then the intact cell binding assay was performed at 4°C. The binding properties of [3H]mepyramine, a cell-penetrating radioligand for H1 receptors, were not changed by histamine pretreatment. Displacement curves for sedative H1-receptor antagonists (diphenhydramine, chlorpheniramine, promethazine, ketotifen, azelastine and oxatomide) against [3H]mepyramine binding were not changed by histamine pretreatment. In contrast, the displacement curves for non-sedative H1-receptor antagonists (mequitazine, bepotastine, olopatadine, epinastine, carebastine, desloratadine and fexofenadine) were changed by histamine pretreatment: two types of changes, i.e. a rightward shift in the monophasic curve or an increase in the proportion of the low affinity component of the biphasic curve, were prevented under hypertonic conditions, in which clathrin-mediated receptor internalization is known to be inhibited. Thus, internalization-mediated changes in the binding properties of H1-receptor antagonists were well correlated with their sedative and nonsedative behaviors, which might confirm their permeability through the biomembrane and possibly the blood brain barrier.

A new murine model of allergic rhinitis by repeated intranasal Cry j 1 challenge

To evaluate the long-lasting effects of new therapeutic approaches to allergies, we established a new model of allergic rhinitis by repeated challenges with intranasal Cry j 1, a common Japanese cedar (Cryptomeria japonica) pollen allergen, in B10.S mice. We sensitized B10.S mice subcutaneously with Cry j 1/alum three times at 1-week intervals. Five weeks after the final sensitization, we challenged the mice by instilling Cry j 1 intranasally for 5 consecutive days starting 1 day after intranasal histamine pretreatment (challenge-1). We challenged the mice by instilling histamine and Cry j 1 intranasally again 12 weeks later (challenge-2). There were significantly more sneezes after challenge-2 than challenge-1. Cry j 1-specific IgE levels in serum were significantly increased in both challenge-1 and 2 after continuous nasal antigen challenge. Serum levels of anti-Cry j 1 IgE in challenge-2 was 2.3 times higher than after challenge-1. Thus, we have established a new model of seasonal allergic rhinitis in B10.S mice by repeated intranasal antigen challenge, and this model may help elucidate mechanisms of allergic rhinitis and the development of new drugs.

Establishment of an allergic rhinitis model in mice for the evaluation of nasal symptoms

The purpose of our study was to establish a new model of allergic rhinitis in mice, eliciting symptoms such as sneezing, infiltration of eosinophils into the nasal mucosa, and antigen-specific IgE production. One of the major human T-cell epitopes in Cry j 1, an allergen of Japanese cedar pollen, is also a major murine T-cell epitope in B10.S mice. Thus we tried to establish an allergic rhinitis model in B10.S mice with Cry j 1 as the antigen. We sensitized B10.S mice subcutaneously with Cry j 1/alum three times at intervals of 1 week. Five weeks after the final sensitization, we challenged the mice by instilling Cry j 1 intranasally from the day after intranasal histamine pretreatment. Soon after, we counted the number of sneezes. We then evaluated the infiltration of eosinophils into the nasal tissues and also measured the serum levels of antigen-specific IgE antibody. In addition, we confirmed the effects of ketotifen fumarate and dexamethasone hydrochloride on these animals. In Cry j 1-sensitized B10.S mice, sneezes, eosinophil peroxidase (EPO) activity in nasal tissues, and Cry j 1-specific IgE clearly increased after intranasal histamine pretreatment and 5 days of continuous intranasal Cry j 1 challenge. Both ketotifen and dexamethasone inhibited the increase in sneezing, and dexamethasone also inhibited EPO activity and Cry j 1-specific IgE. Thus we succeeded in establishing a new model of allergic rhinitis in Cry j 1-sensitized B10.S mice, which exhibited sneezing, eosinophil infiltration into the nasal mucosa, and Cry j 1-specific IgE production.

Highly efficient adenovirus-mediated gene transfer to cardiac myocytes after single-pass coronary delivery.

Efficient and homogeneous gene transfer to cardiac myocytes is a major target in myocardial gene therapy. The aim of this study was to determine the conditions permitting efficient, homogeneous, adenovirus-mediated gene transfer to cardiac myocytes, with a view to application during coronary artery catheterization. Gene transfer to adult rat ventricular myocytes was conducted using type 5 adenoviruses carrying the lacZ reporter gene. Adenovirus delivery via coronary arteries was performed on isolated perfused rat hearts, and gene transfer efficiency was analyzed on whole ventricles, freshly isolated myocytes, and cultured myocytes. Single-pass delivery of 1 X 10(9) PFU associated with 1 min of no-flow yielded only 1 +/- 0.5% of positive myocytes. Pretreatment by histamine perfusion (10(-5) M final concentration) increased this value to 30 +/- 9% (p < 0.001), and pretreatment by Ca2+-free buffer perfusion increased it to 67 +/- 8% (p < 0.001). Combination of the two pretreatments had no additional effect. Increasing the viral dose to 3 X 10(9) PFU increased transfection efficiency only in permeabilized vessels. The 1-min no-flow period after adenovirus delivery was crucial for efficient gene transfer: despite histamine pretreatment, only 2 +/- 1% positive myocytes were observed without flow interruption (p < 0.05 versus 1 min of no-flow). Gene transfer was shown to occur in situ during cardiac perfusion, rather than during heart digestion or myocyte isolation. This study shows that highly efficient adenovirus-mediated gene transfer to cardiac myocytes in situ can be achieved by single-pass intracoronary vector delivery, provided that vascular permeability is first increased and coronary flow is briefly interrupted.

Minor role of a Ca2+-depleted sarcoplasmic reticulum in heterologous desensitization of smooth muscle to K+.

Exposure of porcine carotid artery smooth muscle (PCASM) to histamine was followed by a large reduction in the rate of force generation in response to 40 mM KCl. This was shown to be a manifestation of slow attainment of a steady-state myoplasmic Ca2+ concentration ([Ca2+]i). We hypothesized that if net transsarcolemmal Ca2+ flux into the depolarized PCASM cells is the same before and after a desensitizing histamine treatment, then the transient attenuation of the increase in [Ca2+]i may be due to accelerated uptake of Ca2+ by a partially depleted sarcoplasmic reticulum (SR) acting as a Ca2+ sink or superficial buffer barrier. We tested this hypothesis by eliciting responses of "desensitized PCASM" to 40 mM KCl in the presence of cyclopiazonic acid (CPA), an SR Ca2+-ATPase inhibitor. Contractions of CPA-treated tissues were attenuated less than those of tissues not treated with CPA, but they were not abolished. CPA-insensitive mechanism(s) dominated the desensitization. We conclude that histamine pretreatment reduced net transsarcolemmal Ca2+ flux into PCASM in response to 40 mM KCl.

Regulation of H1-receptor coupling and H1-receptor mRNA by histamine in bovine tracheal smooth muscle.

1. Pretreatment of bovine tracheal smooth muscle (BTSM) with histamine (1-100 microM, 1 h) induced a concentration-dependent desensitization of the contractile response to subsequently administered histamine, with a reduction of the maximum response of 72 +/- 8% (n = 5) following pre-exposure to 100 microM histamine. In contrast, concentration-response curves to the muscarinic agonist, methacholine were not affected following histamine pretreatment, indicating a homologous desensitization. Furthermore, concentration-response curves to NaF, a G-protein activator, were not altered following histamine pre-incubation. 2. The histamine H1-receptor (H1R) desensitization could be antagonized by mepyramine (an H1-receptor antagonist, 1 microM) but not by cimetidine (an H2-receptor antagonist, 10 microM), indicating that the desensitization occurred via stimulation of histamine H1-receptors, without evidence for the involvement of histamine H2-receptors. 3. Indomethacin (10 microM) did not block the H1R desensitization, suggesting no involvement of prostaglandins. Furthermore, histamine pre-incubation in calcium free medium still induced a functional uncoupling of H1R. 4. GF 109203X, a protein kinase C (PKC) inhibitor, and H-7, a non-selective kinase inhibitor, did not antagonize the homologous H1R desensitization. 5. The steady-state level of H1R mRNA, assessed by Northern blot analysis, was not affected by prolonged histamine exposure (100 microM, 0.5, 1, 2, 4, 16 and 24 h). 6. These results suggest that histamine induces desensitization of the H1R at the level of the receptor protein, which involves a mechanism independent of PKC, PKA, PKG and calcium influx, suggesting the involvement of a receptor-specific kinase.

Mast cell mediated ion transport in intestine from patients with and without inflammatory bowel disease

Background—Mast cells have been shown to regulate intestinal ion transport in animal models and normal human colon but their physiological role in human intestinal inflammatory disorders is unknown.Aims—To examine mast...

Heterologous desensitization of smooth muscle to K+ depolarization: retarded stimulus-[Ca2+]i coupling

To understand the phenomenon of postreceptor heterologous desensitization, we exposed porcine carotid media to 40 mM KCl physiological saline solution both before and after intervening treatment with histamine. Increasing histamine concentration or duration of exposure or decreasing the interval between histamine exposure and KCl progressively slowed the contractile responses to K+ depolarization. A delay in initiation and a slower rate of rise of KCl-induced stress in histamine-pretreated muscle were preceded by a slower rate of rise of aequorin-estimated myoplasmic Ca2+ concentration ([Ca2+]i), myosin regulatory light chain (MRLC) phosphorylation, and tissue stiffness, with no detectable change in the Ca2+ sensitivity of MRLC phosphorylation. This heterologous desensitization was not a diminished steady-state force but instead a profound slowing of contraction rates. This slowing was a manifestation of retardation of the rate at which [Ca2+]i rises to the level appropriate for the stimulus. The lack of rapid initial [Ca2+]i and cross-bridge phosphorylation transients as a consequence of histamine pretreatment resulted in very slow cross-bridge cycling rates and rates of force development (latch).

The histamine H1 receptor in GT1-7 neuronal cells is regulated by calcium influx and KN-62, a putative inhibitor of calcium/calmodulin protein kinase II.

1. In GT1-7 cells, histamine stimulated the initial [Ca2+]i transient in a dose-dependent manner with a best-fit EC50 value of 4.2 +/- 4.2 microM (mean +/- s.e.mean, n = 4) and a best-fit maximal effect of 138 +/- 56 nM (n = 4) increase above basal calcium levels. 2. Pretreatment of cells with 30 microM histamine for 30 min desensitized the population mean peak calcium signal by 53% to 75 +/- 9 nM, (n = 3, P < 0.04). Analysis of the individual cells revealed that 39 +/- 7% (n = 94 cells from 8 experiments) of pretreated cells exhibited desensitized histamine-stimulated [Ca2+]i transients of < or = 1 standard deviation below the control cells mean calcium transient level. 3. The desensitization induced by histamine was prevented (P < 0.01) by KN-62 (10 microM), a putative inhibitor of the calcium/calmodulin-dependent protein kinase II (CaMKII). KN-62 (10 microM) alone did not induce [Ca2+]i mobilization, nor did it antagonize the histamine-stimulated [Ca2+]i signal. In addition, KN-62 did not appear to have its effect by hastening the rate of recovery from desensitization. 4. Histamine pretreatment in nominal (zero calcium + 0.2 mM EGTA) or in low (0.3 mM) extracellular calcium did not induce histamine receptor desensitization, supporting a role for extracellular calcium in the homologous H1 receptor desensitization process. 5. Histamine (30 microM) stimulated at least four different types of [Ca2+]i signals in GT1-7 cells. The majority (61%) were of single spikes with the remaining cells showing some form of calcium oscillatory behaviour. The proportion of GT1-7 cells showing histamine-induced calcium oscillations was histamine concentration-dependent and significantly reduced after acute desensitization. KN-62, when present during histamine pretreatment, prevented this fall in calcium oscillation. Under the conditions of nominal or 0.3 mM extracellular calcium the proportion of cells exhibiting histamine-stimulated calcium oscillations was not significantly different from the controls. 6. Bradykinin stimulated a [Ca2+]i transient in GT1-7 cells with a population mean peak response of 147 +/- 8 nM (n = 5) over basal levels. The bradykinin-induced [Ca2+]i signal was without any calcium oscillatory activity. Histamine pretreatment caused the heterologous desensitization of the bradykinin [Ca2+]i signal (44% reduction, P < 0.007), which was unaffected by KN-62. 7. The results presented here suggest that the histamine-mediated homologous H1 receptor desensitization process involves extracellular calcium and can be blocked by KN-62, a putative inhibitor of CaMKII. In contrast, KN-62 does not appear to prevent the histamine-mediated heterologous desensitization cascade. These findings suggest fundamental differences in the mechanisms underlying homologous and heterologous H1 receptor desensitization pathways in GT1-7 neuronal cells.

Characteristics of the binding of [3H]-mepyramine to intact human U373 MG astrocytoma cells: evidence for histamine-induced H1-receptor internalisation.

1. The kinetics of the binding of 5 nM [3H]-mepyramine to sites on intact human U373 MG astrocytoma cells, sensitive to inhibition by 2 microM pirdonium, were temperature-dependent. At 37 degrees C the half-time for association was 0.9 +/- 0.4 min and at 4 degrees C 19 +/- 3 min. Dissociation of bound [3H]-mepyramine was fast at 37 degrees C, t0.5 1.5 +/- 0.3 min, but at 6 degrees C dissociation initiated by dilution or addition of unlabelled mepyramine was negligible over 120 min. The very slow dissociation at 6 degrees C made it possible to reduce the level of pirdonium-insensitive binding from 56 +/- 5% to 39 +/- 5% by washing the cells in ice-cold medium before filtration. 2. The binding of [3H]-mepyramine sensitive to 2 microM temelastine, measured after 10 min equilibration at 37 degrees C, failed to saturate and was resolved into an hyperbola and an apparently linear component, whereas the fit to the binding of [3H]-mepyramine sensitive to 2 microM pirdonium was not significantly improved over that to an hyperbola. The mean Kd for the binding of [3H]-mepyramine to the saturable component, 2.5 +/- 0.4 nM, was in close agreement with the value of 3.5 nM for mepyramine derived from inhibition of histamine H1-receptor-mediated inositol phosphate formation in U373 MG cells. 3. Curves for the inhibition of the binding of 5 nM [3H]-mepyramine to U373 MG cells by histamine H1-receptor antagonists were biphasic and were fitted to a two site-model. Affinities calculated from the best-fit IC50 values for the high-affinity site correlated well with those expected for binding to H1-receptors. 4. The percentages of the high-affinity site in curves of the inhibition of [3H]-mepyramine binding to intact U373 MG cells by two tertiary amine antagonists, norpirdonium and 4-methyldiphenhydramine, 68 +/- 3 and 63 +/- 4%, were significantly greater than the percentages of the high-affinity site in the inhibition curves of their quaternary derivatives, 50 +/- 1 and 45 +/- 3%, respectively. Similarly, the percentage of the high-affinity site for unlabelled mepyramine, 65 +/- 7%, was greater than for the non-cell penetrant H1-antagonist temelastine, 42 +/- 5%. 5. Incubation of U373 MG cells with 100 microM histamine at 37 degrees C, followed by washing twice in ice-cold medium and then incubation with 1-15 nM [3H]-mepyramine for 120 min at 4 degrees C, resulted in a decrease in the binding of [3H]-mepyramine sensitive to 2 microM pirdonium, compared to control cells not exposed to histamine. The binding of [3H]-mepyramine in the absence of pirdonium was not altered by histamine pretreatment, whereas the level of the pirdonium-insensitive binding was significantly increased, except after 1 min exposure to histamine. The decreases in the pirdonium-sensitive binding after 5, 10 and 60 min incubation with 100 microM histamine were 41 +/- 6, 56 +/- 6 and 67 +/- 8%, respectively, but the decrease after 1 min incubation with histamine, 16 +/- 8%, was not statistically significant. 6. The results are consistent with the binding of [3H]-mepyramine to intact U373 MG cells being to both plasma membrane and intracellular histamine H1-receptors. The high-affinity binding sensitive to the non-cell penetrant quaternary compounds and to temelastine is thus to plasma membrane H1-receptors. On exposure to 100 microM histamine receptors are translocated to the intracellular pool, since the change in the high-affinity binding of [3H]-mepyramine is primarily in the level of the pirdonium-insensitive binding, rather than in the total binding.

Histamine Antagonizes Serotonin and Growth Factor-induced Mitogen-activated Protein Kinase Activation in Bovine Tracheal Smooth Muscle Cells

We examined the effects of the bronchoconstrictor agonists serotonin (5-hydroxytryptamine; 5-HT) and histamine on mitogen-activated protein (MAP) kinase activation in cultured bovine tracheal myocytes. Kinase renaturation assays demonstrated activation of the 42- and 44-kDa MAP kinases within 2 min of 5-HT exposure. MAP kinase activation was mimicked by alpha-methyl-5-HT and reduced by pretreatment with either phorbol 12,13-dibutyrate or forskolin, suggesting activation of the 5-HT2 receptor, protein kinase C, and Raf-1, respectively. Raf-1 activation was confirmed by measurement of Raf-1 activity, and the requirement of Raf-1 for 5-HT-induced MAP kinase activation was demonstrated by transient transfection of cells with a dominant-negative allele of Raf-1. Histamine pretreatment significantly inhibited 5-HT and insulin-derived growth factor-1-induced MAP kinase activation. Attenuation of MAP kinase activation was reversed by cimetidine, mimicked by forskolin, and accompanied by cAMP accumulation and inhibition of Raf-1, suggesting activation of the H2 receptor and cAMP-dependent protein kinase A. However, histamine treatment inhibited Raf-1 but not MAP kinase activation following treatment with either platelet-derived growth factor or epidermal growth factor, implying a Raf-1-independent MAP kinase activation pathway. In summary, our data suggest a model whereby 5-HT activates MAP kinase via a protein kinase C/Raf-1 pathway, and histamine attenuates MAP kinase activation by serotonin via activation of cAMP-dependent protein kinase A and inhibition of Raf-1.

Agonist‐induced desensitization of histamine Hi receptor‐mediated inositol phospholipid hydrolysis in human umbilical vein endothelial cells

1. The regulation of histamine-induced [3H]-inositol phosphate formation was studied in human cultured umbilical vein endothelial cells (HUVEC). 2. Histamine (EC50 4.8 microM) produced a 12.7 fold increase in [3H]-inositol phosphate formation over basal levels. Prior exposure to 0.1 mM histamine (2 h) produced a 78% reduction in the response to subsequent histamine (0.1 mM) challenge. The IC50 for this histamine-induced desensitization was 0.9 microM. 3. The inositol phosphate response to histamine (0.1 mM) was inhibited by phorbol dibutyrate (IC50 40 nM; maximal reduction 64%). This effect was antagonized by both staurosporine (100 nM) and Ro 31-8220 (10 microM). However, the histamine-induced desensitization of the H1-receptor-mediated inositol phosphate response was insensitive to the protein kinase inhibitors, staurosporine, Ro 31-8220, K252a and KN62. 4. Prior exposure to sodium nitroprusside (100 microM), forskolin (10 microM) or dibutyryl cyclic AMP (1 mM) had no effect upon histamine-induced [3H]-inositol phosphate formation. 5. NaF (20 mM) and thrombin (EC50 0.4 u ml-1) also induced inositol phosphate formation in HUVEC. Histamine pretreatment (0.1 mM, 10-120 min) failed to modify the inositol phosphate response to a subsequent NaF or thrombin challenge. 6. We conclude that the desensitization of histamine H1-receptor-mediated [3H]-inositol phosphate formation occurs at the level of the receptor and involves a mechanism independent of activation of protein kinase A, G, or C, or calcium calmodulin-dependent protein kinase II.

An immortalised murine hypothalamic neuronal cell, GT1-7, expresses functional histamine H 1 receptors

Histamine, acting via H 1 receptors, dose-dependently stimulated 3H]inositol phosphate production in GT1-7 neuronal cells. GT1-7 cells also responded to Substance P but not to other neuroactive drugs tested. Acute histamine pretreatment desensitised the histamine-induced response, resulting in a reduction in the maximal response and a slower time-course of [ 3H]-inositol phosphate production. The desensitisation phenomenon was reversible, with full recovery by 2 h.

Homologous desensitization of histamine H2 receptors in the human gastric carcinoma cell line MKN-45

The poorly differentiated human gastric carcinoma cell line MKN-45 possesses histamine H2 receptors, which are linked to stimulation of adenosine 3',5'-cyclic monophosphate (cAMP) production. Pretreatment of MKN-45 cells with histamine elicited a rapid and dose-dependent inhibition of cAMP production in response to subsequent administration of histamine, whereas pretreatment with prostaglandin E2 (PGE2) or forskolin, both of which stimulate cAMP formation in these cells, had no effect on the histamine-induced cAMP production. Neither NaF- nor forskolin-induced cAMP production was affected by histamine pretreatment. Scatchard analysis of data obtained for [3H]-tiotidine binding showed that histamine pretreatment affected neither the number of histamine H2 receptors nor the binding affinity of the ligand for the receptors. Finally, histamine pretreatment had no effect on cAMP response to subsequent addition of PGE2, which was reduced by PGE2 pretreatment. These results suggest that histamine induces homologous desensitization of H2 receptors on MKN-45 cells by a mechanism that does not involve stimulation of cAMP production.

Desensitization of histamine H1 receptor-mediated inositol phosphate accumulation in guinea pig cerebral cortex slices.

1. Histamine stimulated the production of [3H]-inositol phosphates in untreated (control) guinea-pig cerebral cortex slices with a best-fit EC50 of 17 +/- 4 microM, and a best-fit maximum response of 385 +/- 23% over basal accumulation. 2. Histamine pretreatment desensitized guinea-pig cortex slices to a subsequent challenge with histamine, which was observed as a reduction in the best-fit maximum response to 182 +/- 32% over basal accumulation. 3. The time-course for the histamine-induced production of [3H]-inositol phosphates was approximately linear over 90 min of stimulation in both control and histamine pretreated slices. The rate of production in pretreated slices was significantly slowed compared to control, such that by 90 min of histamine stimulation the desensitized slices produced 2.8 times the basal [3H]-inositol phosphate accumulation compared to 5.3 fold the basal [3H]-inositol phosphate accumulation in the control slices. 4. Displacement of [3H]-mepyramine binding to homogenates of guinea-pig cerebral cortex by mepyramine and histamine revealed that histamine pretreatment did not alter the apparent affinity of the H1 receptor for histamine (control Kd = 6.3 +/- 0.7 microM, desensitized Kd = 7.9 +/- 1.6 microM) or mepyramine (control Kd = 3.4 +/- 0.8 nM, desensitized Kd = 3.4 +/- 1.3 nM), nor was there any reduction in the calculated maximum number of [3H]-mepyramine binding sites (control Bmax = 192 +/- 31 fmol mg-1 protein, desensitized Bmax = 220 +/- 50 fmol mg-1 protein). 5. The histamine-mediated desensitization of response in guinea-pig slices was mediated by the HI receptor subtype, since the attentuated maximum histamine-stimulated [3H]-inositol phosphate accumulation could not be prevented by inclusion of an H2- (ranitidine) and an H3- (thioperamide) receptor antagonist during the pretreatment period.6. The desensitized histamine-stimulated [3H]-inositol phosphate accumulation recovered to 90% of control levels over a period of 150 min after the removal of the conditioning dose of histamine, with a half-time of recovery of about 95 min.