Galanin receptors in the rat gastrointestinal tract

Abstract

Galanin functions are mediated by three distinct G-protein-coupled receptors, galanin receptor 1 (GalR1), GalR2 and GalR3, which activate different intracellular signaling pathways. Here, we quantified mRNA levels of GalR1, GalR2 and GalR3 in the gastrointestinal tract using real time RT-PCR. GalR1 and GalR2 mRNAs were detected in all segments with the highest levels in the large intestine and stomach, respectively. GalR3 mRNA levels were quite low and mostly confined to the colon. We also investigated the effect of galanin 1-16, which has high affinity for GalR1 and GalR2 and low affinity for GalR3 on depolarization-evoked Ca2+ increases in rat cultured myenteric neurons using Ca2+-imaging. Intracellular Ca2+ changes in myenteric neurons were monitored using the Ca2+ sensitive dye, fluo-4, and confocal microscopy. Galanin 1-16 (1 microM) markedly inhibited the K+-evoked Ca2+ increases in myenteric neurons. In summary, the differential distribution of GalRs supports the hypothesis that the complex effects of galanin in the gastrointestinal tract result from the activation of multiple receptor subtypes. Furthermore, this study confirms the presence of functional GalRs and suggests that galanin modulates transmitter release from myenteric neurons through inhibition of voltage-dependent calcium channels involving a G(i/o)-coupled GalR.

Fig. 1

Expression of GalR mRNAs in different regions of the rat gastrointestinal tract: stomach, small intestine and large intestine. Plot of GalR1 mRNA levels is shown in (a), of GalR2 mRNA in (b) and of GalR3 mRNA in (c). The levels of GalR mRNAs were measured by real time RT-PCR. The data acquired from each sample were normalized to those of β-actin. Relative quantities (RQ) of mRNA were analyzed using the comparative Ct method. Each cDNA sample was amplified in triplicate and all data are expressed as the means ± SEM.

Fig. 2

Galanin 1–16 inhibits voltage dependent Ca²⁺ increases in myenteric neurons. (a) Effects of galanin 1–16 on depolarization evoked Ca²⁺ increases in the soma of a myenteric neuron in culture produced by 1 min applications of elevated [K⁺]_o. Example of somatic [Ca²⁺]_i recorded from a cultured myenteric neuron. (b) Bar graph of galanin 1–16 (1 µM) mediated inhibition of the K⁺-evoked Ca²⁺ increase in cultured myenteric neurons.