Radioimmunoassay of carbonic anhydrase VI in saliva and sheep tissues

Abstract

A specific and sensitive radioimmunoassay has been developed for the measurement of the secreted carbonic anhydrase isoenzyme (CA VI) in sheep saliva and tissues. The assay can detect as little as 75 pg of CA VI, and the antibody used does not cross-react with CA II or CA III. The intra-assay variation, measured using a saliva sample, was 3.0%, whereas the inter-assay variation was 10.5%. The concentration of CA VI in parotid saliva from normal, resting sheep was 5.6 +/- 3.0 micrograms.ml-1 (n = 42) or 79.4 +/- 35.7 micrograms.mg of total protein-1. With feeding, the CA VI concentrations increased an average of 6-fold. The secretion rate of CA VI from the vascularly isolated neurotomized parotid gland of the anaesthetized sheep was 0.62 +/- 0.40 micrograms.min-1, compared with a rate of 11.7 +/- 7.8 micrograms.min-1 from the parotid gland of normal conscious sheep. Stimulation of the parotid-gland preparation by the muscarinic agent bethanechol increased the secretion rate to 438 +/- 172 microgram.min-1 (n = 8), and electrical stimulation of the secretomotor Moussu nerve increased CA VI secretion rate to 634 +/- 330 micrograms.min-1 (n = 4). Submandibular saliva from anaesthetized sheep contained 6.9 +/- 2.1 micrograms of CA VI.ml-1 (n = 3). The only tissues found to contain measurable amounts of CA VI were the parotid (6.4 micrograms.mg of protein-1) and submandibular (1.8 micrograms.mg of protein-1) salivary glands. The sublingual salivary gland, kidney, lung, adrenal, brain, skeletal muscle, liver, heart, pancreas, small intestine and cerebrospinal fluid did not have a measurable CA VI content.