Biochemical and immunological studies and assay of rat sublingual mucins

Original studies of rat sublingual mucins raised questions as to the existence of a second mucin species as distinguished by binding to hydroxyapatite. The existence of multiple mucin species is of concern in pharmacological studies of mucous-cell secretion as each species could represent distinct mucous-cell populations that respond differently to secretagogues. Thus a separate hydroxyapatite-bound mucin pool expressed in rat sublingual glands was isolated and characterized. Biochemical comparison of hydroxyapatite-bound mucins to total and hydroxyapatite-unbound sublingual mucins demonstrated no substantial differences in either amino acid and carbohydrate contents or in size distributions. In addition, a radioimmunoassay was developed using antisera prepared previously against unbound mucins. The three mucin pools exhibited equal specificities in displacement of radiolabelled unbound mucin tracer in the radioimmunoassay. Thus, bound and unbound mucins are indistinguishable, both immunologically and in biochemical composition. The radioimmunoassay was then evaluated for use in pharmacological studies of acinar mucous-cell secretion. Measurement by radioimmunoassay of secretion from isolated acini in response to carbachol was concentration-dependent (EC50 approx. 0.3 μM and maximal stimulation at 1 μM carbachol). In immunolocalization studies the antiserum was highly selective for mucous cells, recognized all mucous cells within histological sections, and was localized subcellularly to mucous-cell secretion granules and trans-Golgi, further validating the radioimmunoassay as a method to detect exocrine secretion from the entire pool of acinar mucous cells. Moreover, the radioimmunoassay was compared and found equivalent to an acid-precipitation method to assess relative secretion, suggesting the acid-precipitation method is also valid for pharmacological studies of isolated acini.