administration of exogenous histamine into human nasal airway causes nasal obstruction , rhinorrhea , and sneezing . these effects appear to be mediated by histamine h1 receptor because h1 receptor antagonists abolish histamine - induced nasal symptoms .   to understand the role of histamine on nasal allergy , the information about the localization of histamine h1 receptor is very important . the previous autoradiografic study using h - pyrilamine has demonstrated h1 receptor existed exclusively on the endothelium of vessels . more recently , sanico et al . found that not only vascular endothelial cells but also epithelial cells and nerves expressed histamine h1 receptor on human inferior turbinates by immunohistochemical studies . mucosal hyperreactivity to histamine can be observed in patients with perennial allergic rhinitis , suggesting upregulation of histamine h1 receptor may exist . however , little is known about upregulation of h1 receptor protein in upper airway .   in the present study , western blotting , immunohistochemistry , and rt - pcr analysis for histamine h1 receptor were performed to confirm both mrna and protein expression of the h1 receptor in human nasal mucosa . human inferior turbinates were obtained after turbinectomy from 12 patients with nasal obstruction refractory to medical therapy . informed consent was obtained from all patients and this study was approved by the ethics committee of sapporo medical university . all were nonsmokers , and 6 patients had perennial allergy against mites as defined by questionnaire and cap test ( pharmacia , uppsala , sweden ) . all medications , including antibiotics , were prohibited for at least 3 weeks prior to the study . the nasal mucosal specimens were dissected from the cartilage , and ( 1 ) immediately frozen in liquid nitrogen and stored at 70c for rna and protein extraction for rt - pcr and western blotting , ( 2 ) placed into cold transfer medium ( rpmi 1640 medium ) for epithelial cell and vascular endothelial cell culture , and ( 3 ) fixed in 10% formalin for immunohistochemistry . human nasal vascular endothelial cells ( hnvecs ) were isolated from nasal inferior turbinates according to a previously described protocol   with minor modification . the nasal specimen was cut into 2-mm sections and enzymatically digested using 0.2% collagenase type iv solution ( sigma , st louis , mo , usa ) for 5  min at 37c , washed with mcbd 131 medium ( sigma ) containing 5% fcs and 2  ng / ml vascular endothelial growth factor ( invitrogen co. , carlsbad , ca , usa ) , and placed in collagen type - i - coated 6-well culture plates ( sumitomo bakelite co. ltd . , after 24  hrs , the medium and the tissue pieces were discarded , and the culture plate was washed twice to remove floating cells . fresh medium mcbd 131 medium ( sigma ) containing 5% fcs and 2  ng / ml vascular endothelial growth factor was added , and the cells were cultured in a 5% carbon dioxide humidified atmosphere at 37c . morphologic observations using a phase contrast microscope showed the hnvecs consisted primarily of vascular endothelial cells . more than 95% of the hnvecs showed positive reactions for anti - human cd31 antibody ( dako , denmark ) . human nasal epithelial cells ( hnecs ) were isolated from human nasal mucosa specimens according to a previously described protocol . nasal specimens were rinsed 2 - 3 times with ham 's f-12 medium ( sigma ) supplemented with antibiotics and incubated in 0.1% protease type xiv in ham 's f-12 medium for 16 hours at 4c . after incubation 10% fetal bovine serum was added to neutralize protease activity and epithelial cells were detached by gentle agitation . cell suspensions were filtered through a 60-mesh cell dissociation sieve ( sigma ) and centrifuged at 500  g for 10  min at room temperature . the cell pellet was then resuspended in hormonally defined ham 's f-12 culture medium ( ham 's hd ) . cell suspensions were plated onto collagen type - i - coated 6-well culture plates ( sumitomo bakelite co. ltd . ) in ham 's hd medium and cultured in a 5% carbon dioxide humidified atmosphere at 37c . morphologic observations using a phase contrast microscope showed the hnecs consisted primarily of epithelial cells . more than 95% of the hnecs showed positive reactions for anti - human cytokeratin antibody ( dako , denmark ) . total rna from nasal mucosa was extracted using trisol reagent ( invitrogen , carlsbad , ca , usa ) . some of rna samples ( 1  g/l ) were treated with dnase i ( 10  u/l ) for 30  min to avoid dna contamination following the second rna extraction . rt - pcr was carried out by using a commercial one step rna pcr kit ( takara biomedicals , tokyo , japan ) . rna ( 50  ng ) was reverse - transcribed into cdna by incubating with 5u amv reverse transcriptase xl at 50c for 30  min . after denaturation at 94c for 2  min , cdna were amplified by 40 cycles of pcr ( 94c for 30  s , 60c for 30  s , and 72c for 1  min ) with 5u of amv - optimized taq dna polymerase to amplify genes for h1 receptor . for the histamine h1 receptor , the forward primers ( 116135 ) : 5-ggtctaagacagagttggtgct-3 and the reverse primers ( 593612 ) : 5-atcctcttctgcaacctggtca-3 ( 497-bp fragment ) were used . pcr products were analyzed by agarose electrophoresis in 1.5% gel and visualized by ethidium bromide staining . tissue samples were homogenised in t - per tissue protein extraction reagent ( thermo fisher scientific inc , waltham , ma , usa ) , supplemented with the proteiase inhibitor cocktail ( sigma ) . and the protein extracts were purified by using pageprep advance kit ( thermo fisher scientific inc . ) . protein concentration in the homogenates was determined using bca protein assay kit ( thermo fisher scientific inc . ) . protein extraxts ( 20  ug protein each ) were separatd on a 412% sodium dodecyl sulphate-(sds- ) polyacrylamide gel and blotted onto a polyvinylidene difluoride ( pvdf ) membrane ( invitrogen corporation , carlsbad , ca , usa ) . membranes were then blocked and probed with rabbit anti - human h1 receptor polyclonal antibody against a peptide corresponding to cytoplasmic loop#3 ( between transmembrane 5 and 6 ) of human histamine h1 receptor ( alpha diagnostics international , san antonio , tx , usa ) or rabbit anti - human glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) polyclonal antibody ( santa cruz biotechnology inc , santa cruz , ca , usa ) as internal control . the bands were visualised using a westernbreeeze chromogenic western blot immunodetection kit ( invitrogen ) . to verify band specificity , anti - h1 receptor antibody was also preincubated with a 100 molar excess of blocking peptides ( alpha diagnostics international ) . the ratio of the expression of h1 receptor protein to beta - actin protein was evaluated by densitometry . for immunohistochemistry of histamine h1 receptor , rabbit anti - human h1 receptor polyclonal antibody ( alpha diagnostics international ) was used at 5  g / ml concentration . deparaffinized sections were initially incubated with 3% h2o2 in methanol for 30  min to quench endogenous peroxidase activity . after microwave treatment ( 10 minutes at 500 watt in citrate buffer ) , the sections were incubated in blocking solution ( 10% normal goat serum in pbs ) for 30  min before incubation in primary antibody . then , the sections were incubated with anti - h1 receptor polyclonal antibody for overnight at 4c , washed , and incubated for 30 minutes with envision+ , peroxidase , rabbit ( dako corporation , carpinteria , ca , usa ) which reacts with rabbit immunoglobulins . a further washing in pbs was followed by developing in dab ( dako ) as a chromogen for signal visualization . the slides were counterstained mayer 's haematoxylin and coverslipped using mounting medium . to verify the signal specificity , anti - h1 receptor antibody was also preincubated with 100 molar excess of blocking peptide ( alpha diagnostics international ) . negative controls were obtained by replacing primary antibodies by rabbit immunoglobulin fraction ( dako ) . histamine h1 receptor and gapdh protein levels evaluated by western blot analysis were determined in duplicate and data are presented as mean  sd . the nonparametric mann - whitney u test was used to determine difference in between perennial allergic rhinitis and nonallergic rhinitis . values with p  0.05 were considered to be statistically significant . figure 1 shows the results of rt - pcr for 40 cycles using total rna extracted from human nasal mucosa ( figure 1 , lane 1 ) , primary cultured human nasal vascular endothelial cells ( figure 1 , lane 2 ) , and primary cultured human nasal epithelial cells ( figure 1 , lane 3 ) , which revealed the expression of histamine h1 receptor mrna . these results suggested that the existence h1 receptor mrna in human nasal epithelial cells and vascular endothelial cells . in order to determine the protein size of h1 receptor in human nasal mucosa , h1 receptor expression was confirmed by using western blot analysis . as shown in figure 2 , the expression of single band for h1 receptor protein could be demonstrated ( figure 2 , lane 1 and 2 ) . the band of approximately 56  kda is of the expected size . the specificity of the polyclonal antibody was ascertained by complete neutralization through preincubation of the anti - h1 receptor polyclonal antibody with its respective specific blocking peptide immunogen ( figure 2 , lanes 3 and 4 ) . densitometric quantification of the bands was performed to evaluate the expression levels of h1 receptor on allergic and nonallergic nasal mucosa . the ratio of h1 receptor / gapdh in nasal mucosa from patients with nasal allergy was significantly higher than the ratio found in nasal mucosa obtained from nonallergic rhinitis ( allergic rhinitis : 0.76  0.24 , nonallergic rhinitis : 0.39  0.11 ,  p = 0.03 ; figure 3(b ) ) . the distribution of h1 receptor in nasal mucosa was examined by means of immunohistochemistry . as shown in figure 4(a ) , immunoreactivity for h1 receptor was significantly detected in epithelial cells and vascular endothelial cells . the intensity of the h1 receptor staining was decreased by coincubation with a specific commercial blocking peptide ( figure 4(b ) ) . specificity of the staining was also confirmed by the absence of labeling with normal rabbit immunoglobulin ( figure 4(c ) ) . as shown in figure 5 the blood vessels in superficial area expressed higher level of h1 receptor immunoreactivity than that in deeper area in the nasal mucosa . the human h1 receptor is a 487-amino acid g - coupled protein with 7 transmembrane domains , and molecular mass of h1 receptor is calculated to be 55,781  da . thus , the molecular mass , about 56  kda for the nasal h1 receptor estimated in this study is comparable with those noted in the previous reports . our present western blot analysis revealed that h1 receptor protein was significantly increased in allergic nasal mucosa in comparison with nonallergic nasal mucosa . this phenomenon can be observed in patients with perennial allergic rhinitis , when compared with healthy control subjects . it is therefore , logical to hypothesize that hyperresponsiveness might stem from upregulation of histamine receptors .   in the past , only few studies have been designed to explore the expression of h1 receptor in nasal mucosa biopsies of patients with allergic rhinitis . by using rt - pcr , h1 receptor mrna levels have been found to be increased in nasal mucosa of perennial allergic rhinitis [ 10 , 11 ] , but not in seasonal allergic rhinitis . on the other hand , little work has been done to evaluate the expression of nasal h1 receptor at protein level . examining biopsy specimens from both humans and guinea pigs , could not demonstrate differences in h1 receptor density between allergic rhinitis and controls using the technique of radioligand binding . it is not yet clear why the levels of h1 receptor are elevated in allergic airway . however , several chemical mediators and cytokines including histamine , platelet activating factor , and il-4 [ 14 , 15 ] upregulate h1 receptor levels . so , our data implies that h1 receptor is upregulated in allergic nasal mucosa , playing a role in the pathogenesis in allergic rhinitis . our present immunohistochemical studies and rt - pcr analysis clearly show the expression of histamine h1 receptor on not only nasal vascular endothelial cell but also nasal epithelial cells at mrna and protein level . in 1992 , demonstrated the presence of h1 receptors in the mucosa of human turbniates by using autoradiographic and pharmacologic techniques . in their study , localization of h1 receptors was clear in the endothelium of arterioles , veins , and sinusoids . however , no evidence of binding to radioactive pyrilamine ( mepyramine ) could be generated on the nasal epithelium in their report . as yet , several studies have reported the effects of histamine on airway epithelial cells in vitro . histamine cause releases of cytokines and chemokines from cultured airway epithelial cells [ 16 , 17 ] . in addition , histamine is an important mediator of epithelial barrier function , leading to increases in paracellular permeability [ 18 , 19 ] . the expression of h1 receptor protein was found not only epithelial cells , but also on superficial blood vessels , especially on endothelium of small vessels in lamina propria . the increase in calcium induced by the activation of h1 receptors leads to the production of nitric oxide in vascular tissues , causing vasodilation , an endothelium- and cyclic guanosine monophosphate ( cgmp)-dependent action . the effects on vascular endothelial cells in vivo lead directly to changes in vascular permeability , allowing for leukocyte infiltration and oedema formation [ 21 , 22 ] . in contrast , we could not confirm the h1 receptor expression on sinusoidal vessels , which are believed to regulate mucosal swelling in the nose . based on the location of h1 receptor , histamine may play minor role on the regulation of vascular swelling in nasal mucosa . this suggestion is supported by a small beneficial effect of h1 receptor antagonists on the nasal congestion during nasal allergic responses . using rt - pcr , western blot analysis , and immunohistochemistry , we have confirmed the expression and the distribution of histamin h1 receptor in human nasal mucosa . the expressions of h1 receptor protein were marked in patients with perennial nasal allergy than in patients with nonallergic rhinitis . our findings should be of considerable interest for understanding the role of histamine in upper airway diseases such as allergic rhinitis and nonallergic rhinitis .
in the present study , we have investigated the expression of histamine h1 receptor in human turbinates by rt - pcr , western blotting , and immunohistochemistry . 
 human turbinates were obtained by turbinectomy from 12 patients with nasal obstruction refractory to medical therapy . 
 rt - pcr analysis of total rna extracted from human nasal turbinate , primary cultured human nasal epithelial cells , and nasal vascular endothelial cells demonstrated the expression of histamine h1 receptor mrna . 
 about 56  kda band was detected in human turbinates by western blot analysis using anti - h1 receptor antibody . 
 the expression level of h1 receptor protein was marked in patients with nasal allergy than in patients with nonallergic rhinitis . 
 the immunohistochemical study revealed that epithelial cells and vascular endothelial cells showed intense immunoreactivity for histamine h1 receptor . 
 in addition , the blood vessels in superficial area expressed higher level of h1 receptor immunoreactivity than that in deeper area in the nasal mucosa . 
 these results may have an important clinical implication for understanding the role of histamine h1 receptor on upper airway diseases such as allergic rhinitis and nonallergic rhinitis .