Immunohistochemical study of leucine-enkephalin and its secretory effects in the isolated pig lacrimal gland

Background: In our previous studies immunohistochemical studies have demonstrated the presence of leucine-enkephalin (Leu-Enk) in the intrinsic nerves of the pig lacrimal gland, which are discernible in the interlobular and interacinar areas from where branches that innervate the acinar tissues are sent. Since the intrinsic nerves have been shown to contain Leu-Enk, this study aimed to investigate the secretory effects of this neuropeptide in isolated segments of the pig lacrimal gland and to reconfirm its presence in the neuronal tissue of the lacrimal gland. Methods: Leu-Enk was identified using immunohistochemical techniques, while total protein output was measured in the effluent samples by an automated on-line colorimetric method. The Ca²⁺ and Mg²⁺ concentrations in the effluent samples were determined using an atomic absorbance spectrophotometer. Results: Leu-Enk (10^-12-10^-7 M) evoked marked increases in total protein output from superfused lacrimal gland segments. The secretory effect of Leu-Enk was not blocked by pretreatment of the tissue with atropine but was substantially reduced by a combination of phentolamine and propranolol. The competitive antagonist, naloxone, has no effect on basal protein output, but when combined with Leu-Enk it caused a significant I eduction in total protein output. Combining theophylline (10^-3 M) with Leu-Enk resulted in a marked potentiation of total protein output. In superfused lacrimal gland segments Leu-Enk (10^-8 M) evoked a net efflux of magnesium (Mg²⁺ release) and a net influx of calcium (Ca²⁺ uptake). Conclusion: The results indicated a physiological role for Leu-Enk in the regulation of protein secretion in the pig lacrimal gland.