| Objectives: The present study aims to unravel the presence of functional GHS-R 1a in the innervation pathways of the gastrointestinal tract of the rat, and to further elucidate the role of peripheral ghrelin in the vagal and spinal visceral afferent pathways involved in sensory reception.Material and Methods: In this study we examined the expression of the functional GHS-R 1a in nodose ganglion (NG), dorsal root ganglion (DRG) nurons and satellite glial cells(SGC) of the rat in mRNA level (in situ hybridization) and at protein level (immunocytochemistry). The extrinsic primary afferent visceroceptive DRG and NG neurons were identified with retrograde tracing fluorogold from the stomach and stained for GHS-Rla and CGRP to determine the expression of GHS-R1a. To further investigate the role of ghrelin in the spinal visceral afferent pathway involved in sensory reception, we used a Confocal Laser Scanning Microscope (CLSM) (one image/s) to monitor the cytosolic Ca2+. concentration ([Ca2+]i)of neurons and satellite glial cells (SGC) in freshly isolated dorsal root ganglia (D2-D14; T8-T13) loaded with Fluo-4-AM (10-6 M; 40 min). [Ca2+]i was recorded respectively after application of ghrelin, ghrelin mimic GHRP-6 and ghrelin antagonist D-Lys-GHRP-6).Results:(1) The presence of the ghrelin receptor (GHS-R1a) in nodose ganglion and dorsal root ganglion neurons and SGC was demonstrated at the mRNA level (in situ hybridization). and at the protein level (immunocytochemistry, using double-labeling with CGRP to identify viscerally projecting neurons).(2) GHS-R 1a was also co-localized with CGRP in some neurons in both DRG and NG, indicating the existence of subpopulations of visceral afferents. (3)Some CGRP-expressing GHS-R 1a immunoreacitve neurons were found containing the retrograde labeled FG.(4) Optical Recording of Cytosolic [Ca2+]:About 55% of the neurons (52/95; 18 ganglia) responded to GH-releasing peptide (GHRP-6; 10-6 M; application 30-60s) with a fast (mean lag time 11 s) and transient rise in [Ca2+]. The amplitude of the responses was independent on the concentration of GHRP-6 (10-13 to 10-5 M) and was on average 1.16±0.15 RF (Relative Fluorescence; mean±SD), being 43% of the KCl response. Almost all (91%) SGC responded to GHRP-6, with a lag time of 11 s and amplitude equal to (103±59%; n=98) the response to KCl(1.62±0.38 RF). Sigmoid dose-response relations (10-14 to 10-5 M), based on the % of responding cells, revealed an EC50 of 10-11 M for both neurons and SGC. Interestingly, this EC50 for the peripherally located ganglion is comparable to previously reported effects of ghrelin on spike activity and [Ca2+] of the hypothalamic arcuate nucleus neurons. Application of ghrelin (rat) did evoke responses in the neurons and SGC comparable to those to GHRP-6 (EC50 10-11 M). (Des-octanoyl)-ghrelin (human; range 10-11 to 10-7 M) did not evoke responses.Conclusion: Our results not only demonstrate the expression of GHS-R 1a in vagal afferents but also provide the first and direct morphological evidence of its presence in spinal visceral afferents. Our results further indicate that ghrelin, in concentrations comparable to those reported in plasma (= 10-10 M), has an excitatory effect on both ganglionic cell types. These findings support the notion of a chemosensory role of the SGC and indicate a modulating role of ghrelin in visceral spinal signaling.
|
PDF Full Text Request