Distributional characteristics of lymphatic vessels in normal human nasal mucosa and sinus mucosa

An immunohistochemical staining technique with the D2-40 antibody was undertaken to examine the functional and morphological features of lymphatic networks in tissue sections and whole-mount preparations of normal nasal mucosa and ethmoid sinus mucosa. In normal nasal mucosa, most lymphatic vessels were found in the superficial mucosa beneath the epithelial layer. Some of these vessels were dilated, whereas others were compressed and had a slit-like lumen. Whole-mount preparations revealed the extent of lymphatic vessels in normal ethmoid sinus mucosa. A network of lymphatic vessels was mainly found in the subepithelial layer, where lymphatic vessels represented rich networks, possessing antler-like branches and typical blind ends. However, these lymphatic networks were not arranged in the form of lymphangion chains, with each lymphangion consisting of a contractile compartment and valve. Thus, recognition of the distinctive features of the lymphatic network in normal nasal and sinus mucosa might aid investigations of lymphatic involvement in sinonasal diseases, such as rhinitis, sinusitis, and malignancy. This work was supported by a Grant-in-Aid for Scientific Research from the Communication Disorders Center, Korea University, Korea.     

Histamine H4 receptor expression is elevated in human nasal polyp tissue

Altered histamine metabolism is thought to be involved in the pathomechanism of nasal polyposis characterized by local eosinophil infiltration. The present study was performed to determine whether histamine receptors play a role in the effect of histamine in nasal polyp tissue. The findings suggest that the expression of H1 and H4 receptors is elevated in polyp tissue (p=0.045; p<0.001), while the level of H2 and H3 receptors is not increased significantly. The elevation of H1 and H4 receptors' expression may indicate that the histamine related mechanisms are preferentially mediated through H1 and H4 histamine receptors in the polyp tissue. Simultaneously with increased H4 receptor expression, the concentration of eosinophil cationic protein (ECP) was increased significantly in polyp tissue (p=0.002). One may speculate that the H4 receptor mediated histamine effects have a role in eosinophil accumulation and activation in inflammatory diseases of the nasal and paranasal sinus mucosa, such as nasal polyposis.     

Alteration of mitochondrial DNA sequence and copy number in nasal polyp tissue

This study was designed to investigate the possibility that mtDNA mutations might arise in inflammatory or chronically damaged nasal polyp tissue from 23 patients. Thirteen patients (57%) displayed nasal polyp tissue-specific mtDNA mutations in the hypervariable segment of the control region and cytochrome b gene, which were not found in the corresponding blood cells and/or adjacent normal tissue. Nasal polyp tissue-specific length heteroplasmic mutations were also detected in nucleotide position (np) 303–315 homopolymeric poly C track (39%), np 514–523 CA repeats (17%) and np 16184–16193 poly C track (30%). The average mtDNA copy number was about three times higher in nasal polyp tissue than in the corresponding peripheral blood cells and adjacent non-polyp tissues. The level of reactive oxygen species (ROS) was significantly higher in the nasal polyp tissues compared to those from the corresponding samples. High level of ROS in nasal polyp tissue may contribute to development of mtDNA mutations, which may play a crucial role in the vicious cycle of pathophysiology of nasal polyps.     

The Cystic Fibrosis Transmembrane Conductance Regulator Impedes Proteolytic Stimulation of the Epithelial Na+ Channel

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that prevent its proper folding and trafficking to the apical membrane of epithelial cells. Absence of cAMP-mediated Cl⁻ secretion in CF airways causes poorly hydrated airway surfaces in CF patients, and this condition is exacerbated by excessive Na⁺ absorption. The mechanistic link between missing CFTR and increased Na⁺ absorption in airway epithelia has remained elusive, although substantial evidence implicates hyperactivity of the epithelial Na⁺ channel (ENaC). ENaC is known to be activated by selective endoproteolysis of the extracellular domains of its α- and γ-subunits, and it was recently reported that ENaC and CFTR physically associate in mammalian cells. We confirmed this interaction in oocytes by co-immunoprecipitation and found that ENaC associated with wild-type CFTR was protected from proteolytic cleavage and stimulation of open probability. In contrast, ΔF508 CFTR, the most common mutant protein in CF patients, failed to protect ENaC from proteolytic cleavage and stimulation. In normal airway epithelial cells, ENaC was contained in the anti-CFTR immunoprecipitate. In CF airway epithelial cultures, the proportion of full-length to total α-ENaC protein signal was consistently reduced compared with normal cultures. Our results identify limiting proteolytic cleavage of ENaC as a mechanism by which CFTR down-regulates Na⁺ absorption.     

Allosteric Inhibition of the Epithelial Na+ Channel through Peptide Binding at Peripheral Finger and Thumb Domains

The epithelial Na⁺ channel (ENaC) mediates the rate-limiting step in transepithelial Na⁺ transport in the distal segments of the nephron and in the lung. ENaC subunits are cleaved by proteases, resulting in channel activation due to the release of inhibitory tracts. Peptides derived from these tracts inhibit channel activity. The mechanism by which these intrinsic inhibitory tracts reduce channel activity is unknown, as are the sites where these tracts interact with other residues within the channel. We performed site-directed mutagenesis in large portions of the predicted periphery of the extracellular region of the α subunit and measured the effect of mutations on an 8-residue inhibitory tract-derived peptide. Our data show that the inhibitory peptide likely binds to specific residues within the finger and thumb domains of ENaC. Pairwise interactions between the peptide and the channel were identified by double mutant cycle experiments. Our data suggest that the inhibitory peptide has a specific peptide orientation within its binding site. Extended to the intrinsic inhibitory tract, our data suggest that proteases activate ENaC by removing residues that bind at the finger-thumb domain interface.     

Aquaporins and CFTR in Ocular Epithelial Fluid Transport

Aquaporins (AQPs) and the cystic fibrosis transmembrane conductance regulator (CFTR) provide the molecular routes for transport of water and chloride, respectively, through many epithelial tissues. In ocular epithelia, fluid transport generally involves secondary active chloride transport, which creates the osmotic gradient to drive transepithelial water transport. This review is focused on the role of AQPs and CFTR in water and ion transport across corneal/conjunctival epithelia, corneal endothelium, ciliary epithelium, and retinal pigment epithelium. The potential relevance of water and chloride transport to common disorders of ocular fluid balance is also considered. Recent data suggest AQPs and CFTR as attractive targets for drug development for therapy of keratoconjunctivitis sicca, recurrent corneal erosions, corneal edema, glaucoma, retinal detachment, and retinal ischemia.     

Detection of Mycobacterium leprae in nasal mucosa biopsies by the polymerase chain reaction

Several discoveries about leprosy indicate that Mycobacterium leprae transmission mainly occurs by inhalation, and the nose is major port of entry and exit. The present study evaluated the clinical application of PCR for detection of M. leprae DNA in nasal mucosa biopsies in untreated leprosy patients (52) and their contacts (99) from the State Reference Center in Sanitary Dermatology and Leprosy, Uberlandia, MG, Brazil. PCR detection of a 372-base pair DNA fragment from M. leprae was accomplished in 36 (69.2%) patients, from which 34 (91.9%) of them were multibacillaries. Furthermore, PCR was positive in 3 (16.7%) of 18 slit-skin smear negative, 4 (25.0%) of 16 skin lesion BI negative, 8 (33.3%) of 24 nasal mucosa BI negative patients, and 10 of 99 contacts (10.1%). The presence of bacilli in 10.1% of the contacts may potentially reflect an occult leprosy, and these patients must be accompanied, followed by a chemoprophylaxy treatment. Considering all PCR results against clinical and BI classification of patients and controls, we have found a sensitivity of 69.2%, a specificity of 89.9%, and an accuracy of 82.8%. It has been demonstrated here through PCR of nasal biopsies that the bacillus invades the mucosa, passing through the nasal inferior turbinate to reach peripheral blood. Therefore, the molecular investigation of invasive nasal biopsies by PCR tests has proven to be useful in defining patients of higher risk of transmission and risk-group contacts, which is an important step to reach the World Health Organization objective towards the elimination of leprosy as a public health problem.     

Na self inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases

The regulation of the open probability of the epithelial Na(+) channel (ENaC) by the extracellular concentration of Na(+), a phenomenon called "Na(+) self inhibition," has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na(+) self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na(+) concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q(10)act = 1.5) activation rate and a strongly temperature-dependant (Q(10)inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na(+) self inhibition depended only on the extracellular Na(+) concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na(+) as well as a reduction of Na(+) outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na(+) self inhibition is an intrinsic property of sodium channels resulting from the expression of the alpha, beta, and gamma subunits of human ENaC in Xenopus oocyte. The extracellular Na(+)-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases.     

Flow-sensitive K+-coupled ATP Secretion Modulates Activity of the Epithelial Na+ Channel in the Distal Nephron

The epithelial Na⁺ channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) is under tonic inhibition by a local purinergic signaling system responding to changes in dietary sodium intake. Normal BK_Ca channel function is required for flow-sensitive ATP secretion in the ASDN. We tested here whether ATP secreted through connexin channels in a coupled manner with K⁺ efflux through BK_Ca channels is required for inhibitory purinergic regulation of ENaC in response to increases in sodium intake. Inhibition of connexin channels relieves purinergic inhibition of ENaC. Deletion of the BK-β4 regulatory subunit, which is required for normal BK_Ca channel function and flow-sensitive ATP secretion in the ASDN, suppresses increases in urinary ATP in response to increases in sodium intake. As a consequence, ENaC activity, particularly in the presence of high sodium intake, is inappropriately elevated in BK-β4 null mice. ENaC in BK-β4 null mice, however, responds normally to exogenous ATP, indicating that increases in activity do not result from end-organ resistance but rather from lowered urinary ATP. Consistent with this, disruption of purinergic regulation increases ENaC activity in wild type but not BK-β4 null mice. Consequently, sodium excretion is impaired in BK-β4 null mice. These results demonstrate that the ATP secreted in the ASDN in a BK_Ca channel-dependent manner is physiologically available for purinergic inhibition of ENaC in response to changes in sodium homeostasis. Impaired sodium excretion resulting form loss of normal purinergic regulation of ENaC in BK-β4 null mice likely contributes to their elevated blood pressure.     

Occurrence,distribution and possible role of the regulatory peptide endothelin in the nasal mucosa

Nasal blood flow is finely regulated by local release of neurotransmitters, neuropeptides and other bioactive molecules acting via paracrine mechanisms. We have investigated the occurrence and distribution in human nasal mucosa of endothelin, a potent vasoconstrictor peptide, by immunocytochemistry and the effect of systemic administration of endothelin-1 on vascular perfusion of rabbit nasal mucosa by laser Doppler flowmetry. Endothelin-like immunoreactivity was demonstrated within vascular endothelial cells in both developing and mature human mucosa. Nasal epithelial cells and some connective tissue cells, presumed to be macrophages, also displayed specific immunostaining. In rabbits injected with endothelin-1, a potent and prolonged nasal vasoconstriction was observed. It is suggested that endothelin released locally may participate in the regulation of nasal blood flow via paracrine mechanisms. Since endothelin has growth-promoting actions on several cell types, it is also tentatively proposed that this regulatory peptide may play a role during development of the nose.     

Epithelial Na+ channel delta subunit mediates acid-induced ATP release in the human skin

The amiloride-sensitive epithelial Na⁺ channel (ENaC) regulates Na⁺ homeostasis in cells and across epithelia. Although we described that ENaCδ is a candidate molecule for a pH sensor in the human brain, the physiological and pathological roles of ENaCδ in non-neuronal tissues are still unknown. Here we show a novel physiological function of ENaCδ in peripheral tissues in humans. Expression analyses at the level of mRNA clearly revealed that ENaCδ was abundantly expressed in human epidermis and keratinocytes. In addition, ENaCδ protein was detected in there. In cultured keratinocytes, acidic stress (pH 5.0) evoked ATP release, which was significantly reduced in the presence of 100 μM amiloride or 10 μM benzamil. In conclusion, ENaCδ may be involved in the mechanism underlying pH sensing followed by the regulation of cell viability in the human skin.     

Role of dopamine transporter (DAT) in dopamine transport across the nasal mucosa

Dopamine is a catecholamine neurotransmitter necessary for motor functions. Its deficiency has been observed in several neurological disorders, but replacement of endogenous dopamine via oral or parenteral delivery is limited by poor absorption, rapid metabolism and the inability of dopamine to cross the blood-brain barrier. The intranasal administration of dopamine, however, has resulted in improved central nervous system (CNS) bioavailability compared to that obtained following intravenous delivery. Portions of the nasal mucosa are innervated by olfactory neurons expressing dopamine transporter (DAT) which is responsible for the uptake of dopamine within the central nervous system. The objective of these studies was to study the role of DAT in dopamine transport across the bovine olfactory and nasal respiratory mucosa. Western blotting studies demonstrated the expression of DAT and immunohistochemistry revealed its epithelial and submucosal localization within the nasal mucosa. Bidirectional transport studies over a 0.1-1 mM dopamine concentration range were carried out in the mucosal-submucosal and submucosal-mucosal directions to quantify DAT activity, and additional transport studies investigating the ability of GBR 12909, a DAT inhibitor, to decrease dopamine transport were conducted. Dopamine transport in the mucosal-submucosal direction was saturable and was decreased in the presence of GBR 12909. These studies demonstrate the activity of DAT in the nasal mucosa and provide evidence that DAT-mediated dopamine uptake plays a role in the absorption and distribution of dopamine following intranasal administration.     

Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft

The epithelial Na⁺ channel (ENaC) has a key role in the regulation of extracellular fluid volume and blood pressure. ENaC belongs to a family of ion channels that sense the external environment. These channels have large extracellular regions that are thought to interact with environmental cues, such as Na⁺, Cl⁻, protons, proteases, and shear stress, which modulate gating behavior. We sought to determine the molecular mechanism by which ENaC senses high external Na⁺ concentrations, resulting in an inhibition of channel activity. Both our structural model of an ENaC α subunit and the resolved structure of an acid-sensing ion channel (ASIC1) have conserved acidic pockets in the periphery of the extracellular region of the channel. We hypothesized that these acidic pockets host inhibitory allosteric Na⁺ binding sites. Through site-directed mutagenesis targeting the acidic pocket, we modified the inhibitory response to external Na⁺. Mutations at selected sites altered the cation inhibitory preference to favor Li⁺ or K⁺ rather than Na⁺. Channel activity was reduced in response to restraining movement within this region by cross-linking structures across the acidic pocket. Our results suggest that residues within the acidic pocket form an allosteric effector binding site for Na⁺. Our study supports the hypothesis that an acidic cleft is a key ligand binding locus for ENaC and perhaps other members of the ENaC/degenerin family.     

Experimental and numerical determination of odorant solubility in nasal and olfactory mucosa

Odorant deposition in the nasal and olfactory mucosas is dependent on a number of factors including local air/odorant flow distribution patterns, odorant mucosal solubility and odorant diffusive transport in the mucosa. Although many of these factors are difficult to measure, mucosal solubility in the bullfrog mucus has been experimentally determined for a few odorants. In the present study an experimental procedure was combined with computational fluid dynamic (CFD) techniques to further describe some of the factors that govern odorant mucosal deposition. The fraction of odorant absorbed by the nasal mucosa (eta) was experimentally determined for a number of odorants by measuring the concentration drop between odorant 'blown' into one nostril and that exiting the contralateral nostril while the subject performed a velopharyngeal closure. Odorant concentrations were measured with a photoionization detector. Odorants were delivered to the nostrils at flow rates of 3.33 and 10 l/min. The velopharyngeal closure nasal air/odorant flows were then simulated using CFD techniques in a 3-D anatomically accurate human nose modeland the mucosal odorant uptake was numerically calculated. The comparison between the numerical simulations and the experimental results lead to an estimation of the human mucosal odorant solubility and the mucosal effective diffusive transport resistance. The results of the study suggest that the increase in diffusive resistance of the mucosal layer over that of a thin layer of water seemed to be general and non-odorant-specific; however, the mucosa solubility was odorant specific and usually followed the trend that odorants with lower water solubility were more soluble in the mucosa than would be predicted from water solubility alone. The ability of this approach to model odorant movement in the nasal cavity was evaluated by comparison of the model output with known values of odorant mucosa solubility.     

Metabolism-dependent activation and toxicity of chemicals in nasal glands

The mucosae of the nasal passages contain a large amount of glands which express secretory proteins as well as phase I and phase II biotransformation enzymes. In this review the metabolic activation, covalent binding and toxicity of chemicals in the Bowman's glands in the olfactory mucosa, in the sero-mucous glands in the nasal septum and in the lateral nasal glands and maxillary glands around the maxillary sinuses are discussed. Light microscopic autoradiographic studies have demonstrated a selective covalent binding of nasal toxicants and carcinogens such as halogenated hydrocarbons and N-nitrosamines, especially in the Bowman's glands following a single systemic exposure, suggesting a high rate of metabolic activation of chemicals in these glands. Special attention is put on the herbicide dichlobenil which induces necrosis in the olfactory mucosa following a cytochrome-P450-mediated metabolic activation and covalent binding in the Bowman's glands.     

The frontal sinus in ancient and modern Greenlandic Inuit

The purpose of this study was to compare the frontal sinus size of ancient Greenlandic Inuit with ancient Inuit of Alaska and Canada, and to compare sinus size between ancient and modern Greenlandic Inuit. Also, it was analyzed whether cranial size was a determinant of frontal sinus size. Frontal sinus size was evaluated in terms of absence frequency and planimetrically. Absence was defined as a frontal sinus not exceeding a line drawn between the supraorbital rims. A significant increase in absence frequency was noted from Alaska over Canada to Greenland (males: p<0.03; females p<0.0001). This is in accordance with earlier studies, indicating that although these Inuit populations once have been commonly related to the Old Bering Sea population, the Greenland Inuit represent an endpoint in an eastward migration. There was a significant increase (p<0.0001) in frontal sinus size from ancient to modern Greenlandic Inuit, probably indicative of a high degree of admixture with non-Inuit after modern colonization. The results regarding craniofacial size parameters and frontal sinus side were inconclusive. No single craniofacial variable showed significant effect on frontal sinus size, but the area displayed sexual dimorphism, females having smaller frontal sinuses.