Galanin inhibition of voltage-dependent Ca(2+) influx in rat cultured myenteric neurons is mediated by galanin receptor 1

Abstract

Galanin activates three receptors, the galanin receptor 1 (GalR1), GalR2, and GalR3. In the gastrointestinal tract, GalR1 mediates the galanin inhibition of cholinergic transmission to the longitudinal muscle and reduction of peristalsis efficiency in the small intestine. Galanin has also been shown to inhibit depolarization-evoked Ca2+ increases in cultured myenteric neurons. Because GalR1 immunoreactivity is localized to cholinergic myenteric neurons, we hypothesized that this inhibitory action of galanin on myenteric neurons is mediated by GalR1. We investigated the effect of galanin 1-16, which has high affinity for GalR1 and GalR2, in the presence or absence of the selective GalR1 antagonist, RWJ-57408, and of galanin 2-11, which has high affinity for GalR2 and GalR3, on Ca2+ influx through voltage-dependent Ca2+ channels in cultured myenteric neurons. Myenteric neurons were loaded with fluo-4 and depolarized by high K+ concentration to activate voltage-dependent Ca2+ channels. Intracellular Ca2+ levels were quantified with confocal microscopy. Galanin 1-16 (0.01-1 microM) inhibited the depolarization-evoked Ca2+ increase in a dose-dependent manner with an EC(50) of 0.172 microM. The selective GalR1 antagonist, RWJ-57408 (10 microM), blocked the galanin 1-16 (1 microM)-mediated inhibition of voltage-dependent Ca2+ channel. By contrast, the GalR2/GalR3 agonist, galanin 2-11 did not affect the K+-evoked Ca2+ influx in myenteric neurons. GalR1 immunoreactivity was localized solely to myenteric neurons in culture, as previously observed in intact tissue. These findings indicate that the inhibition of depolarization-evoked Ca2+ influx in myenteric neurons in culture is mediated by GalR1 and confirm the presence of functional GalR1 in the myenteric plexus. This is consonant with the hypothesis that GalR1 mediates galanin inhibition of transmitter release from myenteric neurons.

Fig. 1

Primary cell culture preparation from rat longitudinal muscle-myenteric plexus (5 days in vitro). A: DIC image of primary cultured myenteric neurons showing cells with long processes. B–C: Simultaneous visualization of MAP2 (green, B and C, neuronal marker) and GFAP (red, B, marker of glial) or actin (red, C, marker of smooth muscle cells) immunoreactivities showing the presence of different types of cells in the preparation. D–F: Primary cultured rat myenteric neurons immunolabeled with MAP2 and GalR1. The same cell is visualized with MAP2 immunoreactivity (D), and GalR1 immunoreactivity (E). F: Simultaneous visualization of MAP2 and GalR1 immunoreactivity. GalR1 immunoreactivity is predominantly localized at the cell surface, whereas MAP2 immunoreactivity is in the cytoplasm. Scale bars: 10 microns.

Fig. 2

Galanin 1–16 inhibition of depolarization-evoked Ca²⁺ increases in a cultured myenteric neuron. A: 1–6 are pseudocolor images corresponding to fluorescence peaks from the same cell plotted in B. 1, control [K⁺]_o (5 mM) mammalian superfusate; 2, elevated [K⁺]_o (72 mM) mammalian superfusate; 3, galanin 1–16 (1 µM) containing mammalian superfusate; 4, galanin 1–16 (1 µM) and elevated [K⁺]_o (72 mM) mammalian superfusate; 5, control [K⁺]_o (5 mM) mammalian superfusate; 6, elevated [K⁺]_o (72 mM) mammalian superfusate. B. A plot of the [Ca²⁺]_i changes in the soma of a cultured myenteric neuron loaded with fluo-4/AM. Each number represents a time-point when an image was captured during the experiment. All measurements were made from regions of interest drawn over the soma (see Materials and Methods). The stimulus for depolarization was a 1 min application of mammalian superfusate containing 72 mM KCl. Scale bar is 10 microns.

Fig. 3

Concentration-dependent inhibition of K⁺-evoked [Ca²⁺]_i increase in cultured myenteric neurons by galanin 1–16. Fluo-4-loaded myenteric neurons were stimulated with elevated [K⁺]_o in the presence of different concentration of galanin 1–16 (0.01–1 µM). Inhibition of K⁺-evoked change produced by galanin 1–16 was normalized to the average of the initial control and subsequent recovery following the test solution. Each point represents the mean ± SE (n=8–17). *P<0.05 compared with control.

Fig. 4

GalR1 antagonist blocks galanin 1–16 inhibition of K⁺-evoked [Ca²⁺]_i increases in rat cultured myenteric neurons. Depolarization-evoked [Ca²⁺]_i increases in myenteric neurons were stimulated by 1-min applications of 72 mM [K⁺]_o mammalian superfusate with galanin 1–16 (1 µM), and galanin 1–16 (1 µM) plus RWJ-57408 (10 µM) or galanin 2–11 (1 µM). The fractional response represents the K⁺-evoked [Ca²⁺]_i increase induced by galanin 1–16 with or without RWJ-57408 or by galanin 2–11 divided by the average of the K⁺-evoked [Ca²⁺]_i increases obtained in basal conditions before and after application of the test substances. Galanin 1–16 alone inhibited the K⁺-evoked [Ca²⁺]_i increase (0.35 ± 0.10, *P=0.0025, n=5). Galanin 1–16 (1 µM) plus the GalR1 antagonist, RWJ-57408 (10 µM) did not inhibit the K⁺-evoked [Ca²⁺]_i increase (1.12 ± 0.10, n=11; **, P < 0.0001, unpaired t-test). Galanin 2–11 (1 µM) did not modify the depolarization-evoked [Ca²⁺]_i increases (n=5). Each bar represents the mean ± SE.