Internalization of the neurokinin 1 receptor in rat myenteric neurons

Abstract

Immunoreactivity for the neurokinin 1 receptor is contained in nerve cell bodies that have been deduced to be intrinsic primary afferent neurons in the myenteric plexus of the rat ileum. This study shows that neurokinin 1 receptor immunoreactivity on these neurons represents receptors that can bind agonist and undergo endocytosis, and explores the properties of that endocytosis. Segments of rat ileum were incubated in Hanks' balanced salt solution for 1 h at 4°C, followed by 1 h at 37°C in physiological saline solution with nicardipine and tetrodotoxin, in the presence or absence of substance P. Tissue was then fixed and whole-mount preparations were processed for fluorescence immunohistochemistry, using antibodies raised against the C-terminus of the neurokinin 1 receptor. The intracellular and surface distributions of receptor immunoreactivity were analysed using confocal microscopy and quantified by computer analysis. In tissue not exposed to substance P, most neurokinin 1 receptor immunoreactivity was confined to the surfaces of nerve cells, and 29% was intracellular. Exogenous substance P (10 −6 M) caused an increase in the amount of intracellular receptor to 72%. This internalization was concentration dependent, and maximum receptor internalization was achieved between 10 −6 M and 10 −5 M substance P ( ec 50=4.9±1.6×10 −7 M). The specific neurokinin 1 receptor antagonist, SR104333 (10 −6 M), inhibited substance P-induced endocytosis. In tissue that was incubated in 5×10 −5 M monensin (to trap endocytosed receptor in the cell), without the addition of substance P, a high level of intracellular neurokinin 1 receptor immunoreactivity (81%) was also present. We deduce that endocytosis in the presence of monensin was stimulated by the release of tachykinins from intrinsic nerve endings, based on the following evidence: when endogenous release of tachykinin was blocked using a high magnesium/low calcium solution, or binding of tachykinins to the receptor was prevented using 10 −6 M SR140333, the intracellular receptor immunoreactivity remained at approximately 40%. Incubation with hypertonic sucrose also trapped receptors on the cell surface. Use of these protocols that modify receptor trafficking showed that agonist induced the neurokinin 1 receptors to aggregate, accumulate in endocytotic vesicles, move to perinuclear organelles and recycle to the surface in less than 1 h. This study indicates that there is sufficient release of endogenous tachykinins in the rat ileum to cause receptor internalization and implies that these intrinsic primary afferent neurons are likely to be under continuous influence from tachykinins in the normal intestine.