The Effects of Autonomic Drugs on the Concentration of Kallikrein-like Proteases and Cysteine-proteinase Inhibitor (Cystatin) in Rat Whole Saliva

Abstract

The influence of isoproterenol, phenylephrine, propranolol, and reserpine on the salivary concentration of kallikrein-like proteases and cysteine-proteinase inhibitor (cystatin) was investigated. The protease activities in saliva from treated rats were studied by means of five different chromogenic substrates. In the isoproterenol- and phenylephrine-treated groups, a significant decrease in protease activity was found, compared with the control group. The protease activity of saliva was found to be elevated by about 25-50% after chronic administration of reserpine (0.5 mg/kg). Specific polyclonal antibodies to rat glandular kallikrein and cystatin were utilized to determine the salivary concentrations of these proteins. Results from the use of anti-kallikrein antibodies in Western blot analysis and crossed immuno-electrophoresis indicated that differences observed in the kallikrein-like protease levels of saliva from treated animals were due to altered immunoreactive protein levels. The salivary concentrations of kallikrein and cystatin were measured by direct radio-immunoassays with specific antibodies. The concentration of cystatin in the saliva of normal animals or animals treated with reserpine or propranolol was very low, but was increased about 100-fold in phenylephrine-treated animals and more than 5000-fold in isoproterenol-treated animals. Western blot analysis with antibodies to submandibular gland mucin, glutamine/glutamic-acid-rich protein (GRP), and proline-rich proteins (PRP) were also utilized to compare the effects of autonomic drugs on these salivary proteins. The salivary mucin showed an increase in reactivity and increased mobility in saliva from both isoproterenol- and phenylephrine-treated animals. The PRP-antibody-immunoreactive bands were significantly prominent in saliva from isoproterenol- or phenylephrine-treated rats, whereas GRP-immunoreactive bands of 55 and 43 kDa showed a marked decrease in saliva from isoproterenol-treated animals.