Bert Ph. M. Menco

Objective To investigate the presence of aquaporin (AQP) water channels 1, 2, and 3 in stratified squamous vocal fold epithelium. Design Immunolocalization analysis of excised ovine vocal fold epithelia. Subjects Sheep. Interventions Ovine vocal fold epithelia were prepared for immunoelectron microscopy using primary antibodies directed against AQP-1, AQP-2, and AQP-3. Photographic profiles of epithelium exposed to each antibody were used to calculate the immunogold labeling density of the plasma membrane and cytoplasm. Main Outcome Measures Density of immunolabeling was compared across 3 regions that represent cell layers closest to the glottal lumen for the plasma membrane and cytoplasm, respectively. Results Labeling densities of AQP-1 and AQP-2 were significantly greater for the plasma membrane region of the luminal cells than for deeper cell layers. Cytoplasmic labeling and labeling of circular structures was greatest for cell layers 2 through 5 beneath the vocal fold surface compared with the surface cell layer. Immunogold labeling of AQP-3, an aquaglyceroporin, in vocal fold epithelium was inconclusive. Conclusion Aquaporins 1 and 2, associated with the plasma membrane region of ovine vocal fold epithelial cells, demonstrate the presence of an intrinsic mechanism to permit transcellular water flux in response to osmotic gradients.

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2006

The sequentially activated molecules of olfactory signal-onset are mostly concentrated in the long, thin distal parts of olfactory epithelial receptor cell cilia. Is this also true for molecules of olfactory signal-termination and -regulation? G-protein receptor kinase 3 (GRK3) supposedly aids in signal desensitization at the level of odor receptors, whereas β-arrestin-2, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and phosphodiesterase (PDE) PDE1C2 are thought to do so at the level of the adenylyl cyclase, ACIII. The Na⁺, K⁺-2Cl⁻-cotransporter NKCC1 regulates Cl⁻-channel activity. In an attempt to localize the subcellular sites olfactory signal-termination and -regulation we used four antibodies to GRK3, two to β-arrestin-2, five to CaMKII (one to both the α and β form, and two each specific to CaMKII α and β), two to PDE1C2, and three to Cl⁻-cotransporters. Only antibodies to Cl⁻-cotransporters labeled cytoplasmic compartments of, especially, supporting cells but also those of receptor cells. For all other antibodies, immunoreactivity was mostly restricted to the olfactory epithelial luminal border, confirming light microscopic studies that had shown that antibodies to GRK3, β- arrestin-2, CaMKII, and PDE1C2 labeled this region. Labeling did indeed include receptor cell cilia but occurred in microvilli of neighboring supporting cells as well. Apical parts of microvillous cells that are distinct from supporting cells, and also of ciliated respiratory cells, immunoreacted slightly with most antibodies. When peptides were available, antibody preabsorption with an excess of peptide reduced labeling intensities. Though some of the antibodies did label apices and microvilli of vomeronasal (VNO) supporting cells, none immunoreacted with VNO sensory structures.

Olfactory marker protein (OMP) participates in the olfactory signal transduction pathway. This is evident from the behavioral and electrophysiological deficits of OMP‐null mice, which can be reversed by intranasal infection of olfactory sensory neurons with an OMP‐expressing adenovirus. Bex, brain expressed X‐linked protein, has been identified as a protein that interacts with OMP. We have now further characterized the interaction of OMP and Bex1/2 by in vitro binding assays and by immuno‐coprecipitation experiments. OMP is a 19 kDa protein but these immunoprecipitation studies have revealed the unexpected presence of a 38 kDa band in addition to the expected 19 kDa band. Furthermore, the 38 kDa form was preferentially co‐immunoprecipitated with Bex from cell extracts. In‐gel tryptic digestion, mass spectrometry, and two‐dimensional gel electrophoresis indicate that the 38 kDa protein behaves as a covalently cross‐linked OMP‐homodimer. The 38 kDa band was also identified in western blots of olfactory epithelium demonstrating its presence in vivo. The stabilities and subcellular localizations of the OMP‐monomer and ‐dimer were studied in transfected cells. These results demonstrated that the OMP‐dimer is much less stable than the monomer, and that while the monomer is present both in the nuclear and cytosolic compartments, the dimer is preferentially located in a Triton X‐100 insoluble cytoskeletal fraction. These novel observations led us to hypothesize that regulation of the level of the rapidly turning‐over OMP‐dimer and its interaction with Bex1/2 is critical for OMP function in sensory transduction.

Purpose. To examine age-related changes in the ultrastructure of Bruch’s membrane with quick-freeze/deep-etch (QFDE) and conventional thin-section transmission electron microscopy (TEM). methods. Four eyes from human donors aged 27, 41, 76, and 78 years were preserved within 4 hours of death. Full-thickness tissue blocks from the macula were prepared for TEM or for QFDE. results. Ultrastructure seen by conventional TEM was revealed in greater detail by QFDE. Cholesterol-containing particles (mean diameter, 80 nm) formed a thin densely packed layer external to the basal lamina of the retinal pigment epithelium (RPE) only in older eyes. The mesh size of the RPE basal lamina was smaller than the particles, and it appeared larger in older eyes. QFDE also revealed less decorated collagen fibrils in older eyes. conclusions. The data suggest that the predilection of a extremely thin sublayer of inner Bruch’s membrane for accumulating lipid particles may eventually lead to a confluent lipid wall capable of isolating the retina from its blood supply. If these lipids originate in the retinal pigment epithelium, then they are unlikely to have passed through the basal lamina in this form. The age-related increase in lipid particles corresponds with an age-related increase in hydraulic resistance determined in excised Bruch’s membrane/choroid by others. QFDE will be useful for future modeling studies of Bruch’s membrane transport and to identify those moieties responsible for deleterious age-related transport changes in Bruch’s membrane.

Microvilli of vomeronasal organ (VNO) sensory epithelium receptor cells project into the VNO lumen. This lumen is continuous with the outside environment. Therefore, the microvilli are believed to be the subcellular sites of VNO receptor cells that interact with incoming VNO‐targeted odors, including pheromones. Candidate molecules, which are implicated in VNO signaling cascades, are shown to be present in VNO receptor cells. However, ultrastructural evidence that such molecules are localized within the microvilli is sparse. The present study provides firm evidence that immunoreactivity for several candidate VNO signaling molecules, notably the G‐protein subunits G_iα2 and G_oα, and the transient receptor potential channel 2 (TRP2), is localized prominently and selectively in VNO receptor cell microvilli. Although G_iα2 and G_oα are localized separately in the microvilli of two cell types that are otherwise indistinguishable in their apical and microvillar morphology, the microvilli of both cell types are TRP2(+). VNO topographical distinctions were also apparent. Centrally within the VNO sensory epithelium, the numbers of receptor cells with G_iα2(+) and G_oα(+) microvilli were equal. However, near the sensory/non‐sensory border, cells with G_iα2(+) microvilli predominated. Scattered ciliated cells in this transition zone resembled neither VNO nor main olfactory organ (MO) receptor cells and may represent the same ciliated cells as those found in the non‐sensory part of the VNO. Thus, this study shows that, analogous to the cilia of MO receptor cells, microvilli of VNO receptor cells are enriched selectively in proteins involved putatively in signal transduction. This provides important support for the role of these molecules in VNO signaling. J. Comp. Neurol. 438:468–489, 2001.

Heat shock, or stress, proteins (HSPs) are induced in response to conditions that cause protein denaturation. Activation of cellular stress responses as a protective and survival mechanism is often associated with chemical exposure. One interface between the body and the external environment and chemical or biological agents therein is the olfactory epithelium (OE). To determine whether environmental odorants affect OE HSP expression, rats were exposed to a variety of odorants added to the cage bedding. Odorant exposure led to transient, selective induction of HSP70, HSC70, HSP25, and ubiquitin immunoreactivities (IRs) in supporting cells and subepithelial Bowman's gland acinar cells, two OE non‐neuronal cell populations involved with inhalant biotransformation, detoxification, and maintenance of overall OE integrity. Responses exhibited odor specificity and dose dependency. HSP70 and HSC70 IRs occurred throughout the apical region of supporting cells; ubiquitin IR was confined to a supranuclear cone‐shaped region. Electron microscopic examination confirmed these observations and, additionally, revealed odor‐induced formation of dense vesicular arrays in the cone‐like regions. HSP25 IR occurred throughout the entire supporting cell cytoplasm. In contrast to classical stress responses, in which the entire array of stress proteins is induced, no increases in HSP40 and HSP90 IRs were observed. Extended exposure to higher odorant doses caused prolonged activation of the same HSP subset in the non‐neuronal cells and severe morphological damage in both supporting cells and olfactory receptor neurons (ORNs), suggesting that non‐neuronal cytoprotective stress response mechanisms had been overwhelmed and could no longer adequately maintain OE integrity. Significantly, ORNs showed no stress responses in any of our studies. These findings suggest a novel role for these HSPs in olfaction and, in turn, possible involvement in other normal neurophysiological processes. J. Comp. Neurol. 432:425–439, 2001.

Several studies have indicated that olfactory responses are impeded by amiloride. Therefore, it was of interest to see whether, and if so which, olfactory epithelial cellular compartments have amiloride-sensitive structures. Using ultrastructural methods that involved rapid freezing, freeze-substitution and low temperature embedding of olfactory epithelia, this study shows that, in the rat, this tissue is immunoreactive to antibodies against amiloride sensitive Na⁺-channels. However, microvilli of olfactory supporting cells, as opposed to receptor cilia, contained most of the immunoreactive sites. Apices from which the microvilli sprout and receptor cell dendritic knobs had much less if any of the amiloride-antibody binding sites. Using a direct ligand-binding cytochemical method, this study also confirms earlier ones that showed that olfactory receptor cell cilia have Na⁺,K⁺-ATPase. It is proposed that supporting cell microvilli and the receptor cilia themselves have mechanisms, different but likely complementary, that participate in regulating the salt concentration around the receptor cell cilia. In this way, both structures help to provide the ambient mucous environment for receptor cells to function properly. This regulation of the salt concentration of an ambient fluid environment is a function that the olfactory epithelium shares with cells of transporting epithelia, such as those of kidney.