Aquaporin Pathways and Mucin Secretion of Bowman's Glands Might Protect the Olfactory Mucosa

Chem. Senses 37: 35–46, 2012 doi:10.1093/chemse/bjr063 Advance Access publication July 10, 2011 Aquaporin Pathways and Mucin Secretion of Bowman’s Glands Might Protect the Olfactory Mucosa Tom T. Solbu and Torgeir Holen Department of Anatomy, Institute of Basic Medical Sciences, Sognsvannsveien 9, University of Oslo, 0317 Oslo, Norway Correspondence to be sent to: Torgeir Holen, Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, PO Box 1110, Blindern, 0317 Oslo, Norway. e-mail: Accepted June 9, 2011 The sense of smell is conveyed by the olfactory sensory neurons of the olfactory mucosa. Uniquely for sensory systems, the olfactory neurons directly face the external environment and are thus vulnerable to infections and changes in the airway surface liquid, but the surface liquid production and maintenance is not well understood. Here we show in rats and mice that Bowman’s glands secrete the mucin MUC5AC. Aquaporin-5 was present at the apical face of the olfactory epithelium, completing a water transport pathway to the surface of the epithelium. Immunogold electron microscopy analysis revealed an intricate network of fine Aquaporin-1–positive fibroblast processes that surround Bowman’s glands, whereas deeper blood vessels were unlabeled for Aquaporin-1. Our results show how the olfactory mucosa might be protected against infections and dehydration generally and how neuronal function is protected against ion concentration changes in the airway surface liquid by rapid replacement of water losses through the aquaporin pathways. Key words: airway surface liquid, electron microscopy, olfactory mucosa Introduction The olfactory mucosa with its Bowman’s glands is situated in the dorsal and caudal nasal cavity (Todd and Bowman 1847; Köllicker 1855). Despite a long history of investigation, the functionofBowman’sglandsandseveralaspectsoftheolfactory mucosa liquid secretion and maintenance are still unsettled. The nasal cavity is lined with ciliated respiratory epithelium containing mucus-secreting goblet cells, whereas the respiratory submucosal glands are not open to the immediate local surface (Bojsen-Moller 1964). The olfactory epithelium lacks goblet cells, and only the olfactory neurons have cilia, which are nonmotile. In humans, when olfactory epithelium is gradually lost by age, or infection, also Bowman’s glands are lost or disrupted along with the olfactory epithelium (Nakashima et al. 1984). Published data on the olfactory mucosa of the important rodent animal models mouse and rat are rather scarce, in particular ultrastructural analysis (Frisch 1967; Seifert 1971; Breipohl 1972). The exact molecular identity of Bowman’s glands secretion products have remained unknown, although histochemical studies showed that Bowman’s glands in rats are positive for periodic acid–Schiff (PAS) staining, indicative of neutral glycoproteins (Bojsen-Moller 1964; Katz and Merzel 1977). A more comprehensive study in mice also found evidence for sulfated glycoproteins (Cuschieri and Bannister 1974). The utility of light microscopy analysis is limited by the complex character of olfactory mucosa, with its interwoven tissues of glands, blood vessels, connective tissue cells and large, converging bundles of olfactory sensory neuron axons penetrating into the bone of the cribriform plate. Electron microscopy studies in different species have observed two types of secretory vesicles in Bowman’s glands. Large, electron-lucent vesicles are found in dark cells and smaller, electron-dense vesicles in light cells (Frisch 1967; Seifert 1971; Breipohl 1972). Studies from a variety of animal models indicate that the content of the electron-lucent secretory vesicles are mucous glycoproteins, whereas the electrondense vesicles are proteinaceous and serous (Getchell and Getchell 1992). Mucins, a glycoprotein family that historically have been difficult to isolate and characterize, can now be investigated due to advances in bioinformatics and transgenic techniques. Recently, the mucin gene family has been extensively investigated in the lower respiratory tract in humans (Rose and ª The Author 2011. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: Abstract 36 T.T. Solbu and T. Holen Tissue preparation and antibody staining for light microscopy Rats and mice were perfusion fixated with 4% paraformaldehyde (PFA). The nasal region was dissected and post-fixed over night in 4% PFA and then decalcinated for 24 h in 10% formic acid. Tissues were then cryoprotected in 30% sucrose before sectioning with a Leica CM3050 S cryostat. Immunoperoxidase and immunofluorescence staining was carried out with the biotin–streptavidin–peroxidase/DAB system and immunofluorescence-coupled secondary antibodies, respectively. All pictures were acquired using Zeiss LSM PASCAL Axioplan 2 Imaging confocal microscope. Antibodies The antibodies are summarized in Table 1. Affinity-purified rabbit AQP1 antibody was a gift from Soren Nielsen (Nielsen et al. 1993). The AQP3 antibody was purchased from Sigma (cat no.: A0303, lot no. 048k1363). An AQP4 antibody from Santa Cruz Biotechnology (SC9888) was used for light microscopy. For electron microscopy, two AQP4 antibodies were used (cat no. A5971, lot no. 116K1630, Sigma; and LS-C3805, lot no. 7091355, LifeSpan Biosciences). An AQP5 antibody was purchased from Calbiochem Table 1 Antibody overview Antigen Immunogen Species Source AQP1 Purified protein Rabbit S. Nielsen (Nielsen et al. 1993) AQP3 aa 275–292 (C-terminus) affinity purified Rabbit Sigma A0303 AQP4 C-terminus, affinity purified Goat Santa Cruz SC-9888 AQP4 aa 249–323 (C-terminus) affinity purified Rabbit Sigma A5971 AQP4 aa 280–296, affinity purifie Rabbit Life Sciences LS-C3805 AQP5 17 aa in C-terminus Rabbit Calbiochem #178615 AQP5 RaTM14 (aa 251–265) Rabbit T. Matsuzaki (Ablimit et al. 2006) Materials and methods AQP5 RaTM41 (aa 244–257) Rabbit T. Matsuzaki (Ablimit et al. 2006) Animals and AQP4 knockout animals CFTR N-terminus Goat Santa Cruz SC-8909 Experimental protocols were approved by the Institutional Animal Care and Use Committee and conform to National Institutes of Health guidelines for the care and use of animals. Studies were conducted with male BN rats (Charles River, Germany). Mice homozygous for targeted disruption of the gene encoding AQP4 (Thrane et al. 2011) and control wild-type C57Bl/6 mice were used. Golf Aa 82–381 (C-terminus) monoclonal Mouse Santa Cruz SC-55545 MUC5AC Recombinant Mucin 5AC protein monoclonal Mouse Lab Vision MS-10331-P0 Voynow 2006). The glycoproteins of the mucin family would be promising candidates for the secretory components of Bowman’s glands, but so far mucins have not been characterized in the olfactory mucosa and few mucin antibodies are presently available for rodent models. Strong expansion of mucins (>100-fold) after fast secretion (Kamijo et al. 1 (...truncated)