An on-line method for the measurement of total protein output in biological fluids and secretory tissues after stimulation of intrinsic nerves and identification of neurotransmitters using immunohistochemical techniques

Proteins are essential ingredients of life and thus it is essential to measure the level of proteins in biological fluids and tissue homogenates. Several methods have previously been described in the literature for the estimation of proteins using either certain dyes which bind to specific groups of polypeptide side chains producing a protein dye colour complex, methods involving copper binding to peptide bonds or application of an eosin B red dye. In this study, an on-line automated technique based on the Lowry method has been used to estimate total protein output from the isolated lacrimal segments. This method can also be used to estimate total protein from saliva, or any other biological fluid, tissue homogenates or secretory tissues. The on-line automated method for the estimation of total protein from secretory tissues and biological fluid was designed mainly to obtain a rapid, simple and consistent graphical interpretation of result within 40-50 min of starting the experiment. The original chart recording of the time- course response can also be used for publication purposes. With this method, it is possible to investigate the effect of electrical field stimulation on the intrinsic secretomotor nerves employing either wire or platinum electrodes embedded in the perfusing chamber. Moreover, the tissue can also be stimulated with different concentrations of either drugs, hormones or neurotransmitters for different time periods. This method can also be combined with morphology whereby the stimulated tissue can be processed for neuropeptide or neurotransmitter immunohistochemistry to determine which neurotransmitters or neuropeptides are involved in the physiological responses. The automated method is simple and rapid and moreover, it can estimate accurately and directly at physiological pH small amounts (ng-μg) of proteins in effluent samples depending on the sensitivity of the chart recorder. In this study, neuropeptides and neurotransmitters were used as secretagogues in addition to electrical field stimulation.