Expression and localization of endothelin receptors: implications for the involvement of peripheral glia in nociception

Abstract

The endothelins (ETs) are peptides that have a diverse array of functions mediated by two receptor subtypes, the endothelin A receptor (ET(A)R) and the endothelin B receptor (ET(B)R). Pharmacological studies have suggested that in peripheral tissues, ET(A)R expression may play a role in signaling acute or neuropathic pain, whereas ET(B)R expression may be involved in the transmission of chronic inflammatory pain. To begin to define the mechanisms by which ET can drive nociceptive signaling, autoradiography and immunohistochemistry were used to examine the distribution of ET(A)R and ET(B)R in dorsal root ganglia (DRG) and peripheral nerve of the rat, rabbit, and monkey. In DRG and peripheral nerve, ET(A)R-immunoreactivity was present in a subset of small-sized peptidergic and nonpeptidergic sensory neurons and their axons and to a lesser extent in a subset of medium-sized sensory neurons. However, ET(B)R-immunoreactivity was not seen in DRG neurons or axons but rather in DRG satellite cells and nonmyelinating ensheathing Schwann cells. Thus, when ETs are released in peripheral tissues, they could act directly on ET(A)R-expressing sensory neurons and on ET(B)R-expressing DRG satellite cells or nonmyelinating Schwann cells. These data indicate that ETs can have direct, nociceptive effects on the peripheral sensory nervous system and that peripheral glia may be directly involved in signaling nociceptive events in peripheral tissues.

Fig. 1.

¹²⁵I-ET-1-binding sites (A) in monkey DRG are most dense in ring-like structures that surround Nissl-stained DRG neuronal cell bodies (B) identified byasterisks, indicating that endothelin receptors are present on DRG satellite cells. However, ¹²⁵I-ET-1-binding sites are also seen over Nissl-stained small-diameter DRG neuronal cell bodies (denoted by arrowheads), indicating that a subpopulation of neurons express endothelin receptors. Scale bar, 50 μm.

Fig. 2.

DRG satellite cells express ET_BR. A single section of rabbit DRG was double labeled for ET_BR (shown in red; A) and for GFAP (shown ingreen; B). In DRG, the vast majority of ET_BR-immunoreactivity colocalized with GFAP-immunoreactivity but was absent from DRG neuronal cell bodies, indicating that ET_BR is expressed primarily by peripheral DRG satellite cells. Images are single confocal optical sections. Scale bar, 50 μm.

Fig. 3.

ET_AR is expressed primarily by small-diameter neurons of the rat DRG. A–C, Single sections through rat DRG were double labeled for ET_AR (red; A) and NeuN, a marker of all neuronal cell bodies (green; B), to reveal colocalization of the two markers (yellow; C). D–F,A single section through rat DRG double labeled for ET_AR (red; D) and CGRP, a marker of unmyelinated primary afferent neurons (green; E), to reveal colocalization indicated that approximately half of DRG neurons that were immunoreactive for ET_AR were also immunoreactive for CGRP (yellow; F).G–I, Conversely, when single sections were double labeled for ET_AR (red; G) and RT-97, a marker of myelinated primary afferent neurons (green; H), the incidence of colocalization was much lower (I). Scale bars: A–C, 50 μm; D–I, 75 μm.

Fig. 4.

¹²⁵I-Endothelin-1-binding sites in sciatic nerve indicate that endothelin receptors are expressed by ensheathing Schwann cells. ¹²⁵I-Endothelin-1-binding sites show a close correspondence with GFAP-immunoreactivity. A, B, Serial sections processed for ¹²⁵I-ET-1 binding (A) or for GFAP-immunoreactivity (B) suggest that the vast majority of endothelin receptors are located on peripheral supporting cells such as ensheathing (nonmyelinating) Schwann cells. Arrowheadsindicate areas of overlap between ¹²⁵I-ET-1-binding sites and GFAP-immunoreactivity. C, As further evidence that¹²⁵I-ET-1-binding sites are present on peripheral supporting cells, ¹²⁵I-ET-1-binding sites accumulate on all sides of two tight ligatures placed ∼1 cm apart in rat sciatic nerve. The accumulation of binding sites that was seen between the two ligatures indicates that a significant number of these binding sites originate in peripheral supporting cells. P indicates the aspect of the nerve proximal to the dorsal root ganglion;D indicates the distal aspect. Scale bars: A, B, 100 μm; C, 200 μm.

Fig. 5.

In rat sciatic nerve, ET_BRs are localized to ensheathing Schwann cells, whereas ET_ARs are present on primary afferent fibers. A–C, Single sections of rat sciatic nerve were double labeled for ET_BR (red; A) and GFAP (green; B) and visualized with confocal microscopy to determine colocalization as indicated byyellow in C. D–F, Conversely, sections that were double labeled for ET_AR (red; D) and CGRP, a marker of primary afferent fibers (green; E), showed colocalization as demonstrated by yellow inF. Scale bar, 200 μm.