Bronchiolar nonciliated secretory (Clara) cells: source of guanylin in the mammalian lung.

Abstract

The peptide guanylin, which has recently been isolated from the intestine, is involved in the regulation of fluid secretion in the intestinal epithelium by activation of guanylate cyclase C, the putative guanylin receptor. Since the latter protein is also expressed in airway epithelia, we investigated the lung of three mammalian species for the presence and cellular localization of guanylin by immunoblot (Western blot) analyses and light and electron microscopical immunocytochemistry. In Western blots of bovine, guinea pig, and rat lung extracts, three different guanylin antisera directed against the midportion and against the C terminus of the precursor molecule identified a peptide band corresponding to the apparent molecular mass of guanylin. Localization studies in the lung revealed that guanylin is exclusively confined to nonciliated secretory (Clara) cells in the lining of distal conducting airways. The presence of guanylin in the lung and particularly its specific localization to Clara cells indicate that these cells may play a pivotal role in the local (paracrine) regulation of electrolyte/water transport in airway epithelia.

Clara cell impact in air-side activation of CFTR in small pulmonary airways

“…Lung tissues from rat and man (obtained during surgical resection) were separated from large airways by microdissection and prepared for reverse transcription (RT)-PCR analyses as described (13) Peptides and Abs. Immunogenic peptides were synthesized as Cterminal amides by using a standard fluorenylmethoxycarbonyl protocol and used for immunization of rabbits as described (13,16,17). The antisera generated and used here were K605 and K606, each directed against proguanylin-(101-115), K39 directed against proguanylin-(25-37), K42 directed against proguanylin-(34-46), K735 recognizing GC-C-(31-45) and K741 recognizing GC-C-(1009-1023), EG (1)-PKIIA directed against cGKII-(146-158), EG (2)-PKIIB directed against cGKII-(734-749), and EG (1)-AE2 and EG (2)-AE2 both directed against AE2-(1214-1236), EG (1)-NHE-RF1 recognizing EBP50͞NHERF-(295-306), and EG (1)-NHE-RF2 recognizing E3KARP͞NHERF-2-(274-285).…”

Expression of guanylin in “pars tuberalis-specific cells” and gonadotrophs of rat adenohypophysis

“…Two region-specific rabbit antisera against guanylin that have been raised against guanylin-(34-46) (antiserum K42) and guanylin-(101-115) (antiserum K605) were used as detailed (8); both antisera have been characterized and used in previous studies (8,13). Moreover, a panel of antisera was employed for identification and characterization of guanylin immunoreactive cells: anti-human growth hormone (lot AFP872189); anti-rat prolactin (lot AFPC2381); anti-human luteinizing hormone (h-␤-LH, lot AFP54372); anti-human thyroid stimulating hormone (h-␤-TSH, lot AFP62423473); and anti-human chorionic gonadotropin (lot AFP310784)-all from the National Hormone and Pituitary Program of The National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases (Baltimore, MD).…”

“…The homogenates were centrifuged at 20,000 ϫ g for 20 min at 4°C and extracted by applying them to an octadecasilyl (C18) SepPak cartridge (Waters) as described (8). The tissue extracts were separated by Tricine-SDS͞PAGE (21) in 16.5% polyacrylamide gels and electroblotted onto hydrophobic poly(vinylidene difluoride)-based membranes (Pall) as described (8,13). The membranes were incubated overnight at 4°C with the guanylin antisera K42 and K605 (see below), each in a 1:1,500 dilution.…”

“…Although the function of lymphoguanylin remains to be clarified in detail, guanylin and uroguanylin are obviously involved in electrolyte͞water secretion in various ion-transporting epithelia (6). Guanylin and uroguanylin have been localized in paracrine͞luminocrine-secreting epithelial cells of the gastrointestinal tract (3,(7)(8)(9)(10)(11)(12) and of the small airways (13). On the other hand, a circulating form of guanylin in considerable concentrations has been identified in plasma (14), indicating that endocrine cells and organs may be another source of guanylin.…”

Natriuretic peptides: potential modulators of circulatory, renal, respiratory, digestive, endocrine, and central nerve systems and implications of their role in bone remodeling

“…Histochemically, guanylin is exclusively confined to the nonciliated cells (Clara cells) in the lining of distal conducting airways. The presence of guanylin in the Clara cells indicate that the cells may play a pivotal role in the paracrine regulation of electrolyte/water transport in airway epithelia (85) (Fig. 2).…”