Effects of histamine on human periodontal ligament fibroblasts under simulated orthodontic pressure.

As type-I-allergies show an increasing prevalence in the general populace, orthodontic patients may also be affected by histamine release during treatment. Human periodontal ligament fibroblasts (PDLF) are regulators of orthodontic tooth movement. However, the impact of histamine on PDLF in this regard is unknown. Therefore PDLF were incubated without or with an orthodontic compressive force of 2g/cm2 with and without additional histamine. To assess the role of histamine-1-receptor (H1R) H1R-antagonist cetirizine was used. Expression of histamine receptors and important mediators of orthodontic tooth movement were investigated. PDLF expressed histamine receptors H1R, H2R and H4R, but not H3R. Histamine increased the expression of H1R, H2R and H4R as well as of interleukin-6, cyclooxygenase-2, and prostaglandin-E2 secretion even without pressure application and induced receptor activator of NF-kB ligand (RANKL) protein expression with unchanged osteoprotegerin secretion. These effects were not observed in presence of H1R antagonist cetirizine. By expressing histamine receptors, PDLF seem to be able to respond to fluctuating histamine levels in the periodontal tissue. Increased histamine concentration was associated with enhanced expression of proinflammatory mediators and RANKL, suggesting an inductive effect of histamine on PDLF-mediated osteoclastogenesis and orthodontic tooth movement. Since cetirizine inhibited these effects, they seem to be mainly mediated via histamine receptor H1R.

Store-operated calcium entry (SOCE) contributes to phosphorylation of p38 MAPK and suppression of TNF-α signalling in the intestinal epithelial cells.

Calcium influx via store-operated calcium entry (SOCE) has an important role for regulation of vast majority of cellular physiological events. MAPK signalling is also another pivotal modulator of many cellular functions. However, the relationship between SOCE and MAPK is not well understood. In this study, we elucidated the involvement of SOCE in Gαq/11 protein-mediated activation of p38 MAPK in an intestinal epithelial cell line HT-29/B6. In this cell line, we previously showed that the stimulation of M3 muscarinic acetylcholine receptor (M3-mAChR) but not histamine H1 receptor (H1R) led to phosphorylation of p38 MAPK which suppressed tumor necrosis factor-α (TNF-α)-induced NF-κB signalling through ADAM17 protease-mediated shedding of TNF receptor-1 (TNFR1). First, we found that stimulation of M3-mAChR and protease-activated receptor-2 (PAR-2) but not H1R induced persistent upregulation of cytosolic Ca2+ concentration through SOCE. Activation of M3-mAChR or PAR-2 also suppressed TNF-α-induced NF-κB phosphorylation, which was dependent on the p38 MAPK activity. Time course experiments revealed that M3-mAChR stimulation evoked intracellular Ca2+-dependent early phase p38 MAPK phosphorylation and extracellular Ca2+-dependent later phase p38 MAPK phosphorylation. This later phase p38 MAPK phosphorylation, evoked by M3-mAChRs or PAR-2, was abolished by inhibition of SOCE. Thapsigargin or ionomycin also phosphorylate p38 MAPK by Ca2+ influx through SOCE, leading to suppression of TNF-α-induced NF-κB phosphorylation. Finally, we showed that p38 MAPK was essential for thapsigargin-induced cleavage of TNFR1 and suppression of TNF-α-induced NF-κB phosphorylation. In conclusion, SOCE is important for p38 MAPK phosphorylation and is involved in TNF-α signalling suppression.

Hypothermia in mouse is caused by adenosine A1 and A3 receptor agonists and AMP via three distinct mechanisms.

Small mammals have the ability to enter torpor, a hypothermic, hypometabolic state, allowing impressive energy conservation. Administration of adenosine or adenosine 5'-monophosphate (AMP) can trigger a hypothermic, torpor-like state. We investigated the mechanisms for hypothermia using telemetric monitoring of body temperature in wild type and receptor knock out (Adora1-/-, Adora3-/-) mice. Confirming prior data, stimulation of the A3 adenosine receptor (AR) induced hypothermia via peripheral mast cell degranulation, histamine release, and activation of central histamine H1 receptors. In contrast, A1AR agonists and AMP both acted centrally to cause hypothermia. Commonly used, selective A1AR agonists, including N6-cyclopentyladenosine (CPA), N6-cyclohexyladenosine (CHA), and MRS5474, caused hypothermia via both A1AR and A3AR when given intraperitoneally. Intracerebroventricular dosing, low peripheral doses of Cl-ENBA [(±)-5'-chloro-5'-deoxy-N6-endo-norbornyladenosine], or using Adora3-/- mice allowed selective stimulation of A1AR. AMP-stimulated hypothermia can occur independently of A1AR, A3AR, and mast cells. A1AR and A3AR agonists and AMP cause regulated hypothermia that was characterized by a drop in total energy expenditure, physical inactivity, and preference for cooler environmental temperatures, indicating a reduced body temperature set point. Neither A1AR nor A3AR was required for fasting-induced torpor. A1AR and A3AR agonists and AMP trigger regulated hypothermia via three distinct mechanisms.

Olanzapine-induced hyperglycemia: possible involvement of histaminergic, dopaminergic and adrenergic functions in the central nervous system.

BACKGROUND/AIMS: Atypical antipsychotic drugs such as olanzapine are known to induce metabolic disturbance. We have already shown that olanzapine induces hepatic glucose production through the activation of hypothalamic adenosine 5'-monophosphate-activated protein kinase (AMPK). However, it is unclear how olanzapine activates hypothalamic AMPK. Since olanzapine is known to antagonize several receptors, including histaminergic, muscarinic, serotonergic, dopaminergic and adrenergic receptors, we examined the effect of each receptor antagonist on blood glucose levels in mice. Moreover, we also investigated whether these antagonists activate hypothalamic AMPK. METHODS: Male 6-week-old ICR mice were used. Blood glucose levels were determined by the glucose oxidase method. AMPK expression was measured by Western blotting. RESULTS: Central administration of olanzapine (5-15 nmol i.c.v.) dose-dependently increased blood glucose levels in mice, whereas olanzapine did not change blood insulin levels. Histamine H1 receptor antagonist chlorpheniramine (1-10 µg i.c.v.), dopamine D2 receptor antagonist L-sulpiride (1-10 µg i.c.v.) and α1-adrenoceptor antagonist prazosin (0.3-3 µg i.c.v.) also significantly increased blood glucose levels in mice. In contrast, the blood glucose levels were not affected by muscarinic M1 receptor antagonist dicyclomine (1-10 µg i.c.v.) or serotonin 5-HT2A receptor antagonist M100907 (1-10 ng i.c.v.). Olanzapine-induced hyperglycemia was inhibited by the AMPK inhibitor compound C, and AMPK activator AICAR (10 ng to 1 µg i.c.v.) significantly increased blood glucose levels. Olanzapine (15 nmol), chlorpheniramine (10 µg), L-sulpiride (10 µg) and prazosin (3 µg) significantly increased phosphorylated AMPK in the hypothalamus of mice. CONCLUSION: These results suggest that olanzapine activates hypothalamic AMPK by antagonizing histamine H1 receptors, dopamine D2 receptors and α1-adrenoceptors, which induces hyperglycemia.

Nerve growth factor enhances cough via a central mechanism of action.

The mechanisms involved in enhanced cough induced by central and inhaled NGF in guinea pigs were investigated. Cough and airway function were assessed by plethysmography following inhaled or intracerebroventricular (i.c.v.) NGF treatment. Expression of TrkA and/or TRPV1 was determined in bronchi and/or brainstem by real-time PCR and immunoblotting. I.c.v. and inhaled NGF enhanced citric acid induced-cough and airway obstruction. Pretreatment (i.c.v.) with antagonists of TrkA (K252a) or TRPV1 (IRTX) significantly reduced both the NGF (i.c.v.) enhanced cough and airway obstruction whereas the NK1 antagonist (FK888) inhibited only cough. The H1 antagonist (cetirizine) did not affect either. Inhaled NGF increased phosphorylation of TrkA receptors in the bronchi but not the brainstem at 0.5h post-treatment. TrkA mRNA was elevated at 0.5h in the bronchi and at 24h in the brainstem while TRPV1 mRNA was elevated from 0.5h to 24h in brainstem and at 24h in the bronchi. Pretreatment (i.c.v.) with IRTX, but not K252a, significantly inhibited the inhaled NGF-enhanced cough. Central NGF administration enhances cough and airway obstruction by mechanisms dependent on central activation of TrkA, TRPV1 and NK1 receptors while inhaled NGF enhances cough via a mechanism dependent on central TRPV1 and not TrkA receptors. These data show that NGF, in addition to its effects on the airways, has an important central mechanism of action in the enhancement of cough. Therefore, therapeutic strategies targeting NGF signaling in both the airways and CNS may be more effective in the management of cough.

Investigation on signal transduction pathways after H(1) receptor activated by histamine in C6 glioma cells: involvement of phosphatidylinositol and arachidonic acid metabolisms.

BACKGROUND: Information related to histamine-induced cellular responses in C6 glioma cells through second messenger pathways has not been fully studied, especially the involvement of arachidonic acid (AA) metabolism. In addition, specific labeled ligand binding to histamine receptor sites still needs to be clarified. METHODS: Labeled mepyramine ligand was used to study its binding sites; [(3)H] inositol was used to detect inositol 4-phosphate (IP(1)) formation, and fura-2/AM was used to detect intracellular free calcium ion ([Ca(2+)]i) level activated by the phosphatidylinositol-phospholipase C (PI-PLC) pathway. Also, labeled AA was used to detect the metabolism of AA and its metabolites release via the activation of phospholipase A2 in the presence of histamine. RESULTS: C6 glioma cells incubated with histamine in the presence of 10 mM LiCl for 60 minutes induced an increase of IP(1) and glycerophosphoric-inositol (GPI) accumulation. In addition, histamine caused an increase of extracellular AA with its metabolite release, eliciting a transient and sustained increase of free [Ca(2+)]i. The sustained increase of [Ca(2+)]i was almost or completely blocked by La(3+) and excess ethylene diamine tetraacetic acid. The calcium ion influx associated with the sustained phase required the presence of histamine on the receptor sites, and could be blocked by a H(1) antagonist, chlorpheniramine. CONCLUSION: C6 glioma cells possess histamine H(1) receptors that have affinity towards [(3)H]mepyramine binding, and are coupled to PI-PLC to generate inositol phosphates and to increase [Ca(2+)]i, and they are coupled to phospholipase A2 (PLA2) to generate GPI and AA with its metabolite release. The transient increase in [Ca(2+)]i can be attributed to Ca(2+) release from intracellular stores, whereas the sustained increase in [Ca(2+)]i is due to influx of extracellular calcium ions. The sustained increase in [Ca(2+)]i plays a role in the activation of histamine receptor-coupled PLA2.

Gap junctional communication controls the overall endothelial calcium response to vasoactive agonists.

AIMS: A cytosolic calcium (Ca(2+)(i)) increase is an important activation signal for the endothelium. We investigated whether interendothelial spreading of the Ca(2+) signal via gap junctions (GJs) plays a role for the overall Ca(2+)(i) increase in response to vasoactive agonists. METHODS AND RESULTS: In human umbilical vein endothelial cells (HUVECs), a Ca(2+)(i) increase (Fura2) in response to histamine or ATP occurred initially only in about 30% of the cells (initially responding cells) reflecting the cell fraction expressing H(1) or purinergic receptors (FACS/immunohistochemistry). In the remaining adjacent cells, Ca(2+)(i) increases occurred only after a delay of up to 5 s. Blockade of GJ communication (meclofenamic acid and heptanol, or H(2)O(2); verified by dye injection) did not affect responses in the initially responding cells but abolished the delayed Ca(2+)(i) response of the remaining adjacent cells. The resulting reduction in the global endothelial Ca(2+)(i) response significantly reduced the nitric oxide synthesis (assessed as cGMP levels). Similar Ca(2+)(i) results were obtained in the endothelium of freshly isolated mouse (C57BL/6) aortas stimulated with ATP. The receptor-independent Ca(2+)(i) response to ionomycin occurred simultaneously in all cells, regardless of GJ inhibition. In separate experiments, inhibition of the IP(3) receptor (xestospongin-C; 40, µmol/L) but not of the ryanodine receptor (ryanodine, 250 µmol/L) reduced the spread of the Ca(2+)(i) signal into adjacent cells over longer distances. CONCLUSION: The global Ca(2+)(i) response of the endothelium to agonists is determined decisively by the functionality of GJs, thus establishing a new role for GJs in controlling endothelial activity and vasomotor function.

Histamine differentially regulates the production of Th1 and Th2 chemokines by keratinocytes through histamine H1 receptor.

Histamine is a biological amine that plays an important role in allergic responses. However, the involvement of histamine signaling in late allergic responses in the skin is poorly understood. Therefore, we attempted to investigate the involvement of histamine signaling in late allergic responses, especially in keratinocytes (KCs). HaCaT KCs and normal human KCs (NHKs) predominantly expressed histamine H1 receptor (H1R) and H2 receptor (H2R). Histamine suppressed tumor necrosis factor α (TNF-α)- and interferon-γ (IFN-γ)-induced production of CC chemokine ligand 17(CCL17), a type 2 T-helper (Th2) chemokine, by HaCaT KCs. It suppressed the phosphorylation of p38 mitogen-activated protein (MAP) kinase, but not that of extracellular signal-regulated kinases (ERKs), and TNF-α- and IFN-γ-induced nuclear factor κB (NFκB) activity. In contrast, histamine enhanced the production of CXC chemokine ligand 10 (CXCL10), a Th1 chemokine, by TNF-α- and IFN-γ-stimulated HaCaT KCs and NHKs. TNF-α- and IFN-γ-induced CXCL10 production was upregulated by suppression of p38 MAP kinase or NF-κB activity, which could explain histamine involvement. We concluded that histamine suppresses CCL17 production by KCs by suppressing p38 MAP kinase and NF-κB activity through H1R and may act as a negative-feedback signal for existing Th2-dominant inflammation by suppressing CCL17 and enhancing CXCL10 production.

Molecular signaling of pruritus induced by endothelin-1 in mice.

Endothelin-1 (ET-1) has recently been identified to evoke pruritus/itching sensation in both humans and animals. It is most likely that the signaling is through the specific G-protein-coupled ET(A) and ET(B) receptors, but the downstream signaling mediators for ET-1 remain elusive. In the present study, we examined the potential involvement of several distinct signaling molecules in ET-1-induced pruritus in a murine model. We applied an in vivo pruritus model in C57BL/6J mice by injecting ET-1 intradermally into the scruff, and recording the number of scratching bouts within 30 min after injection. Then specific antagonists/inhibitors for distinct signaling molecules, including cell-surface ET(A) and ET(B) receptors, histamine receptor type 1 (H1 receptor), protein kinases A (PKA) and C (PKC), phospholipase C (PLC) or adenylyl cyclase (AC), were co-injected with ET-1. The results showed that ET-1 induced a vigorous scratching response in mice in a dose-dependent manner. This response was further enhanced by a specific antagonist for ET(B) receptor, BQ-788, reduced by a specific antagonist for ET(A) receptor, BQ-123, and not affected by mepyramine, the specific inhibitor for H1 receptor. In addition, the scratching response was significantly reduced by inhibitors for PKC and AC, but was significantly enhanced by PLC inhibitor, while PKA inhibitors showed no effects in the ET-1-induced scratching response. Our data suggested that ET-1 may signal through the ET(A) receptor, AC and PKC pathway to induce pruritus sensation, while ET(B) receptor and PLC may antagonize the pruritus evoked by ET-1. These results may provide a basis for the future development of antipruritic therapy.

Histaminergic responses by hypothalamic neurons that regulate lordosis and their modulation by estradiol.

How do fluctuations in the level of generalized arousal of the brain affect the performance of specific motivated behaviors, such as sexual behaviors that depend on sexual arousal? A great deal of previous work has provided us with two important starting points in answering this question: (i) that histamine (HA) serves generalized CNS arousal and (ii) that heightened electrical activity of neurons in the ventromedial nucleus of the hypothalamus (VMN) is necessary and sufficient for facilitating the primary female sex behavior in laboratory animals, lordosis behavior. Here we used patch clamp recording technology to analyze HA effects on VMN neuronal activity. The results show that HA acting through H1 receptors (H1R) depolarizes these neurons. Further, acute administration of estradiol, an estrogen necessary for lordosis behavior to occur, heightens this effect. Hyperpolarization, which tends to decrease excitability and enhance inhibition, was not affected by acute estradiol or mediated by H1R but was mediated by other HA receptor subtypes, H2 and H3. Sampling of mRNA from individual VMN neurons showed colocalization of expression of H1 receptor mRNA with estrogen receptor (ER)-alpha mRNA but also revealed ER colocalization with the other HA receptor subtypes and colocalization of different subtypes with each other. The latter finding provides the molecular basis for complex "push-pull" regulation of VMN neuronal excitability by HA. Thus, in the simplest causal route, HA, acting on VMN neurons through H1R provides a mechanism by which elevated states of generalized CNS arousal can foster a specific estrogen-dependent, aroused behavior, sexual behavior.

Pouch colon associated with anorectal malformations fails to show spontaneous contractions but responds to acetylcholine and histamine in vitro.

PURPOSE: Congenital pouch colon (CPC) associated with anorectal malformation (ARM) is most commonly reported from Northern India. So far, no physiologic study comparing the detailed contractile status of CPC with non-CPC conditions are available. The present article deals with the contractile study and histopathologic observations in CPC, which may be useful for better surgical management. METHODS: Freshly excised 12 neonatal CPC and similar number of non-CPC (control) specimens were transferred to ice-cold (4 degrees C-6 degrees C) Krebs-Ringer solution bubbled with 100% oxygen. Longitudinally prepared 2 to 4 colonic strips were obtained from central part of each specimen and subjected to the contraction recording after exposure to cumulative concentrations of acetylcholine (ACh) and histamine. Acetylcholine-induced contractions were evaluated after application of atropine (muscarinic blocker), and histaminergic contractions were recorded after pheniramine (H(1) blocker), lignocaine (neuronal blocker), and atropine. Histopathologic observations were made by using H&E and Masson trichrome stains. RESULTS: Control specimens showed spontaneous contractions, but CPC strips did not. Both control and CPC responded to ACh and histamine. The response to histamine was greater (P < .05) in CPC as compared to control, whereas the response to ACh was more (P < .05) in control. In CPC, response of histamine (100 micromol/L) was blocked by pheniramine (0.32 mmol/L) and lignocaine (4 mmol/L) by 97% and 80%, respectively, and enhanced by 57% after preapplication of atropine (10 micromol/L). Acetylcholine (100 micromol/L)-induced contractions were attenuated (86%) in presence of atropine. Histopathologic examination showed fewer mature ganglion cells with various changes in muscle layers including fibrosis, disruption, hypertrophy, atrophy, and constriction bands. CONCLUSION: Congenital pouch colon associated with ARM lacks normal spontaneous contractions but retains ACh and histamine-induced contractility. In view of the functional and histologic abnormalities, we propose that CPC associated with ARM is an abnormally functional and developed tissue. Therefore, resection of the pouch should be considered for better functional outcome of the remaining bowel.

Mast cells down-regulate CD4+CD25+ T regulatory cell suppressor function via histamine H1 receptor interaction.

Mast cells promote both innate and acquired immune responses, but little is known about the effect of mast cells on T regulatory (T(reg)) cell function. In this study, we show for the first time that the capacity of murine CD4(+)CD25(+) T(reg) cells to suppress in vitro proliferation by CD4(+)CD25(-) T responder (T(resp)) cells in response to anti-CD3/anti-CD28 mAb-coated beads was reduced in the presence of syngeneic bone marrow-derived mast cells (BMMC) activated by FcepsilonR cross-linking. Activated BMMC culture supernatants or exogenous histamine also inhibited T(reg) cell suppressor function while the histamine H1 receptor-specific antagonist loratadine, but not the H2 receptor-specific antagonist famotidine, restored T(reg) cell suppressor function in the presence of activated BMMC or activated BMMC culture supernatants. Moreover, treatment of T(reg) cells with loratadine, but not famotidine, rescued T(reg) cell suppressor function in the presence of exogenous histamine. In addition, the H1 receptor-specific agonist 2-pyridylethylamine dihydrochloride inhibited T(reg) cell suppressor function to an extent that was comparable to histamine, whereas the H2 receptor-specific agonist amthamine dihydrobromide was without effect. Both T(reg) cells and T(resp) cells expressed H1 receptors. Exposure to histamine caused T(reg) cells to express lower levels of CD25 and the T(reg) cell-specific transcription factor Foxp3. Taken together, these data indicate that BMMC-elaborated histamine inhibited T(reg) cell suppressor function by signaling through the H1 receptor. We suggest that histamine released as a result of mast cell activation by microbial products might cause a transient decrease in T(reg) cell suppressor function, thereby enhancing the development of protective immunity.

Adenosine in the tuberomammillary nucleus inhibits the histaminergic system via A1 receptors and promotes non-rapid eye movement sleep.

Adenosine has been proposed to promote sleep through A(1) receptors (A(1)R's) and/or A(2A) receptors in the brain. We previously reported that A(2A) receptors mediate the sleep-promoting effect of prostaglandin D(2), an endogenous sleep-inducing substance, and that activation of these receptors induces sleep and blockade of them by caffeine results in wakefulness. On the other hand, A(1)R has been suggested to increase sleep by inhibition of the cholinergic region of the basal forebrain. However, the role and target sites of A(1)R in sleep-wake regulation remained controversial. In this study, immunohistochemistry revealed that A(1)R was expressed in histaminergic neurons of the rat tuberomammillary nucleus (TMN). In vivo microdialysis showed that the histamine release in the frontal cortex was decreased by microinjection into the TMN of N(6)-cyclopentyladenosine (CPA), an A(1)R agonist, adenosine or coformycin, an inhibitor of adenosine deaminase, which catabolizes adenosine to inosine. Bilateral injection of CPA into the rat TMN significantly increased the amount and the delta power density of non-rapid eye movement (non-REM; NREM) sleep but did not affect REM sleep. CPA-promoted sleep was observed in WT mice but not in KO mice for A(1)R or histamine H(1) receptor, indicating that the NREM sleep promoted by A(1)R-specific agonist depended on the histaminergic system. Furthermore, the bilateral injection of adenosine or coformycin into the rat TMN increased NREM sleep, which was completely abolished by coadministration of 1,3-dimethyl-8-cyclopenthylxanthine, a selective A(1)R antagonist. These results indicate that endogenous adenosine in the TMN suppresses the histaminergic system via A(1)R to promote NREM sleep.

Small mouse cholangiocytes proliferate in response to H1 histamine receptor stimulation by activation of the IP3/CaMK I/CREB pathway.

Cholangiopathies are characterized by the heterogeneous proliferation of different-sized cholangiocytes. Large cholangiocytes proliferate by a cAMP-dependent mechanism. The function of small cholangiocytes may depend on the activation of inositol trisphosphate (IP(3))/Ca(2+)-dependent signaling pathways; however, data supporting this speculation are lacking. Four histamine receptors exist (HRH1, HRH2, HRH3, and HRH4). In several cells: 1) activation of HRH1 increases intracellular Ca(2+) concentration levels; and 2) increased [Ca(2+)](i) levels are coupled with calmodulin-dependent stimulation of calmodulin-dependent protein kinase (CaMK) and activation of cAMP-response element binding protein (CREB). HRH1 agonists modulate small cholangiocyte proliferation by activation of IP(3)/Ca(2+)-dependent CaMK/CREB. We evaluated HRH1 expression in cholangiocytes. Small and large cholangiocytes were stimulated with histamine trifluoromethyl toluidide (HTMT dimaleate; HRH1 agonist) for 24-48 h with/without terfenadine, BAPTA/AM, or W7 before measuring proliferation. Expression of CaMK I, II, and IV was evaluated in small and large cholangiocytes. We measured IP(3), Ca(2+) and cAMP levels, phosphorylation of CaMK I, and activation of CREB (in the absence/presence of W7) in small cholangiocytes treated with HTMT dimaleate. CaMK I knockdown was performed in small cholangiocytes stimulated with HTMT dimaleate before measurement of proliferation and CREB activity. Small and large cholangiocytes express HRH1, CaMK I, and CaMK II. Small (but not large) cholangiocytes proliferate in response to HTMT dimaleate and are blocked by terfenadine (HRH1 antagonist), BAPTA/AM, and W7. In small cholangiocytes, HTMT dimaleate increased IP(3)/Ca(2+) levels, CaMK I phosphorylation, and CREB activity. Gene knockdown of CaMK I ablated the effects of HTMT dimaleate on small cholangiocyte proliferation and CREB activation. The IP(3)/Ca(2+)/CaMK I/CREB pathway is important in the regulation of small cholangiocyte function.

Antispasmodic activity of essential oil from Lippia dulcis Trev.

AIM OF THIS STUDY: To investigate the essential oil of Lippia dulcis Trev. (Verbenaceae) that is traditionally used in the treatment of cough, colds, bronchitis, asthma, and colic in Middle America for antispasmodic activity. MATERIALS AND METHODS: We used a porcine bronchial bioassay to study contractile responses to carbachol and histamine in the absence or presence of the essential oil. RESULTS: The essential oil showed anti-histaminergic and anti-cholinergic activities at 100 microg/ml. CONCLUSIONS: The anti-histaminergic and anti-cholinergic activities of the essential oil of Lippia dulcis support the rational use of the plant or plant extracts to treat bronchospasm.

Compromised E-cadherin adhesion and epithelial barrier function with activation of G protein-coupled receptors is rescued by Y-to-F mutations in beta-catenin.

Activation of the type 1 histamine (H1) or the type 2 protease-activated (PAR-2) G protein-coupled receptors interrupts E-cadherin adhesion and decreases the transepithelial resistance (TER) of epithelium. Several reports suggest that cadherin adhesive function depends on the association of cadherin with beta-catenin and that this association is regulated by phosphorylation of tyrosines in beta-catenin. We tested the hypothesis that loss of cadherin adhesion and compromise of TER on activation of the H1 or PAR-2 receptor is due to phosphorylation of tyrosines in beta-catenin. L cells were stably transfected to express E-cadherin (L-E-cad cells) and H1 (L-H1-E-cad cells). L cells and Madin-Darby canine kidney (MDCK) cells constitutively express PAR-2. Stably transfected L-E-cad, L-H1-E-cad, and MDCK cells were also stably transfected with FLAG-tagged wild-type (WT) or mutant beta-catenin, converting tyrosine 142, 489, or 654 to the nonphosphorylatable mimetic, phenylalanine (WT, Y142F, Y489F, or Y654F). Activation of H1 or PAR-2 interrupted adhesion to an immobilized E-cadherin-Fc fusion protein of L-H1-E-cad, L-E-cad, and MDCK cells expressing WT or Y142F beta-catenin but did not interrupt adhesion of L-H1-E-cad, L-E-cad, and MDCK cells expressing the Y489F or Y654F mutant beta-catenins. PAR-2 activation decreased the TER of monolayers of MDCK cells expressing WT or Y142F beta-catenin 40-45%. However, PAR-2 activation did not decrease the TER of monolayers of MDCK cells expressing Y489F or Y654F beta-catenin. The protein tyrosine phosphatase PTP1B binds to the cadherin cytoplasmic domain and dephosphorylates beta-catenin. Inhibition of PTP1B interrupted adhesion to E-cadherin-Fc of MDCK cells expressing WT beta-catenin but did not affect the adhesion of MDCK cells expressing Y489F or Y654F beta-catenin. Similarly, inhibition of PTP1B compromised the TER of MDCK cells expressing WT beta-catenin but did not affect the TER of MDCK cells expressing Y489F or Y654F beta-catenin. We conclude that phosphorylation of tyrosines 489 and 654 in beta-catenin is a necessary step in the process by which G protein-coupled H1 and PAR-2 receptors interrupt E-cadherin adhesion. We also conclude that activation of PAR-2 has no effect on the TER without first interrupting E-cadherin adhesion.

Histamine H1-receptors differentially mediate the action of amylin on hypothalamic neurons in control and in overweight rats.

The hypothalamic arcuate, dorsomedial and paraventricular nuclei are involved in regulation of body weight and food intake and contain binding sites for the anorexigenic amylin. Effects of amylin on medial arcuate and paraventricular neurons studied in adult rats overweight through early postnatal overfeeding in small litters (SL) differed from those of control litters (CL). Now we observed that also dorsomedial neurons respond differentially to this satiety signal. They were significantly inhibited by amylin in SL but not CL rats. Since the histaminergic system seems to be involved in mediating effects of amylin, we studied the role of histamine H(1)-receptors. Single unit activity was recorded in brain slices of CL and SL rats in each of the three hypothalamic nuclei. The histamine H(1)-receptor antagonist pyrilamine differentially altered or reduced responses to amylin, not depending on the kind of litter but on the functional effect of the peptide. Pyrilamine prevented significant inhibition of medial arcuate neurons in controls as well as inhibition of dorsomedial and paraventricular neurons in SL rats. Searching for further mechanisms possibly contributing to the change of neuronal responses we found that in the presence of a GABA(A)-receptor antagonist amylin induced a significant inhibition of medial arcuate neurons in SL rats similar to that in CL without antagonist. Activation of medial arcuate neurons expressing the orexigenic neuropeptide Y and inhibition of dorsomedial and paraventricular neurons in SL rats may in vivo contribute to hyperphagia and overweight. Histamine H(1)-receptors and GABA(A)-receptors seem to be differentially involved in mediation of these effects.

Histamine receptor H1 and dermatopontin: new downstream targets of the vitamin D receptor.

UNLABELLED: In this study, we used multipotential MSCs and microarray assays to follow the changing patterns of gene expression as MSCs were differentiated to osteoblasts. We analyzed co-expressed gene groups to identify new targets for known transcription factor VDR during differentiation. The roles of two genes (histamine receptor H1 and dermatopontin) as downstream targets for the VDR were confirmed by gel electromotility shift, siRNA inhibition, and chromatin immunoprecipitation assays. INTRODUCTION: Osteogenesis is stringently controlled by osteoblast-specific signaling proteins and transcription factors. Mesenchymal stem or multipotential stromal cells from bone marrow (MSCs) have been shown to differentiate into osteoblasts in the presence of vitamin D(3). MATERIALS AND METHODS: We used MSCs and microarray assays to follow the changing patterns of gene expression as MSCs were differentiated to osteoblasts. The data were analyzed with a previously developed strategy to identify new downstream targets of the vitamin D receptor (VDR), known osteogenesis transcription factor. Hierarchical clustering of the data identified 15 distinct patterns of gene expression. Three genes were selected that expressed in the same time-dependent pattern as osteocalcin, a known target for the VDR: histamine receptor H1 (HRH1), Spondin 2 (SPN), and dermatopontin (DPT). RT-PCR, electromotility shift, siRNA inhibition assays, and chromatin immunoprecipitation assays were used to analyze the role of VDR in activation of DPT and HRH1 during differentiation. RESULTS AND CONCLUSIONS: RT-PCR assays confirmed that the genes were expressed during differentiation of MSCs. The roles of two genes as downstream targets for the VDR were confirmed by gel electromotility shift and chromatin immunoprecipitation assays that showed the presence of VDR complex binding sequences. Overexpression of VDR in MG-63 osteosarcoma cells induced the expression of HRH1 and DPT. Inhibition studies with siRNA to DPT and HRH1 showed a decrease in MSC differentiation to osteogenic lineage. In addition, osteogenic differentiation of MSCs was inhibited by the HRH1 inhibitor mepyramine but not the HRH2 inhibitor ranitidine. In conclusion, we show that analysis of co-expressed gene groups is a good tool to identify new targets for known transcription factors.

Histamine H1 receptor involvement in prepulse inhibition and memory function: relevance for the antipsychotic actions of clozapine.

Histamine H(1) blockade is one of the more prominent actions of the multi-receptor acting antipsychotic clozapine. It is currently not known how much this H(1) antagonism of clozapine contributes to the therapeutic or adverse side effects of clozapine. The current studies with Sprague-Dawley rats were conducted to determine the participation of histaminergic H(1) receptor subtype in sensorimotor plasticity and memory function affected by clozapine using tests of prepulse inhibition (PPI) and radial-arm maze choice accuracy. The PPI impairment caused by the glutamate antagonist dizocilpine (MK-801) was significantly attenuated by clozapine. In the current project, we found that the selective H(1) antagonist pyrilamine also reversed the dizocilpine-induced impairment in PPI of tactile startle with an auditory prepulse. In the radial-arm maze (RAM), pyrilamine, like clozapine, impaired working memory and caused a significant dose-related slowing of response. Pyrilamine, however, decreased the number of reference memory errors. We have previously shown that nicotine effectively attenuates the clozapine-induced working memory impairment, but in the current study, nicotine did not significantly alter the effects of pyrilamine on the RAM. In summary, the therapeutic effect of clozapine in reversing PPI impairment was mimicked by the H(1) antagonist pyrilamine, while pyrilamine had a mixed effect on cognition. Pyrilamine impaired working memory but improved reference memory in rats. Thus, H(1) antagonism seems to play a role in part of the beneficial actions of antipsychotics, such as clozapine.