Expression and distribution of GABA and GABAB-receptor in the rat adrenal gland

Abstract

The inhibitory effects of gamma-aminobutyric acid (GABA) in the central and peripheral nervous systems and the endocrine system are mediated by two different GABA receptors: GABAA-receptor (GABAA-R) and GABAB-receptor (GABAB-R). GABAA-R, but not GABAB-R, has been observed in the rat adrenal gland, where GABA is known to be released. This study sought to determine whether both GABA and GABAB-R are present in the endocrine and neuronal elements of the rat adrenal gland, and to investigate whether GABAB-R may play a role in mediating the effects of GABA in secretory activity of these cells. GABA-immunoreactive nerve fibers were observed in the superficial cortex. Some GABA-immunoreactive nerve fibers were found to be associated with blood vessels. Double-immunostaining revealed GABA-immunoreactive nerve fibers in the cortex were choline acetyltransferase (ChAT)-immunonegative. Some GABA-immunoreactive nerve fibers ran through the cortex toward the medulla. In the medulla, GABA-immunoreactivity was seen in some large ganglion cells, but not in the chromaffin cells. Double-immunostaining also showed GABA-immunoreactive ganglion cells were nitric oxide synthase (NOS)-immunopositive. However, neither immunohistochemistry combined with fluorescent microscopy nor double-immunostaining revealed GABA-immunoreactivity in the noradrenaline cells with blue-white fluorescence or in the adrenaline cells with phenylethanolamine N-methyltransferase (PNMT)-immunoreactivity. Furthermore, GABA-immunoreactive nerve fibers were observed in close contact with ganglion cells, but not chromaffin cells. Double-immunostaining also showed that the GABA-immunoreactive nerve fibers were in close contact with NOS- or neuropeptide tyrosine (NPY)-immunoreactive ganglion cells. A few of the GABA-immunoreactive nerve fibers were ChAT-immunopositive, while most of the GABA-immunoreactive nerve fibers were ChAT-immunonegative. Numerous ChAT-immunoreactive nerve fibers were observed in close contact with the ganglion cells and chromaffin cells in the medulla. The GABAB-R-immunoreactivity was found only in ganglion cells in the medulla and not at all in the cortex. Immunohistochemistry combined with fluorescent microscopy and double-immunostaining showed no GABAB-R-immunoreactivity in noradrenaline cells with blue-white fluorescence or in adrenaline cells with PNMT-immunoreactivity. These immunoreactive ganglion cells were NOS- or NPY-immunopositive on double-immunostaining. These findings suggest that GABA from the intra-adrenal nerve fibers may have an inhibitory effect on the secretory activity of ganglion cells and cortical cells, and on the motility of blood vessels in the rat adrenal gland, mediated by GABA-Rs.

Keywords: GABA; GABAB-receptor; adrenal gland; ganglion cells; rat.

Figure 1

Fluorescent micrograph of gamma-aminobutylic acid (GABA) immunoreactivity in the rat adrenal cortex. GABA-immunoreactive nerve fibers are seen along the blood vessels (V) and among the cortical cells. C, capsule; Co, cortex; V, blood vessel. Scale bar: 30 μm.

Figure 2

Fluorescent micrographs of double-immunostaining with gamma-aminobutyric acid (GABA) (A) and choline acetyltransferase (ChAT) (B) antibodies in the same section of the rat adrenal cortex. GABA-immunoreactive nerve fiber (single arrow) is ChAT-immunonegative, while ChAT-immunoreactive nerve fiber (double arrows) is GABA-immunonegative. Scale bar: 60 μm.

Figure 3

Fluorescent micrographs of double-immunostaining of gamma-aminobutyric acid (GABA) (A) and nitric oxide synthase (NOS) (B) antibodies in the same section of the rat adrenal juxtamedullary cortex. GABA-immunoreactive ganglion cells (asterisks) are NOS-immunopositive. Scale bar: 30 μm.

Figure 4

Fluorescent micrographs of formaldehyde-induced fluorescence (FIF) (A) and immunostaining of gamma-aminobutyric acid (GABA) (B) antibody, double-immunostaining of phenylethanolamine N-methyltransferase (PNMT) (C) and GABA (D) antibodies in the same section (A and B, C and D) of the rat adrenal medulla. A few GABA-immunoreactive nerve fibers (arrows) are found in the medulla (B, D). However, no GABA-immunoreactivity is seen in noradrenaline cells (NA) showing blue-white fluorescence and in adrenaline cells (A) demonstrating PNMT-immunoreactivity (A and B, C and D). Scale bar: 40 μm.

Figure 5

Fluorescent micrographs of double-immunostaining of gamma-aminobutyric acid (GABA) (A) and nitric oxide synthase (NOS) (B) antibodies in the same section of the rat adrenal medulla. Numerous GABA-immunoreactive nerve fibers are in close contact with GABA-immunonegative ganglion cells (A) (asterisks). NOS-immunoreactivity is seen in these ganglion cells (B) (asterisks). Scale bar: 30 μm.

Figure 6

Fluorescent micrographs of double-immunostaining of gamma-aminobutyric acid (GABA) (A) and choline acetyltransferase (ChAT) (B) antibodies in the same section of the rat adrenal medulla. Few GABA-immunoreactive nerve fibers (arrowhead) are ChAT-immunopositive, while some GABA-immunoreactive nerve fibers (arrows) are ChAT-immunonegative. Numerous ChAT-immunoreactive nerve fibers are GABA-immunonegative. Scale bar: 50 μm.

Figure 7

Fluorescent micrograph of gamma-aminobutyric acid_B-receptor (GABA_B-R)-immunoreactivity in the rat adrenal medulla. A cluster of GABA_B-R-immunoreactive ganglion cells is found in the medulla. Scale bar: 50 μm.

Figure 8

Fluorescent micrographs of formaldehyde-induced fluorescence (FIF) (A) and immunostaining of gamma-aminobutyric acid_B-receptor (GABA_B-R) (B) antibody, double-immunostaining of phenylethanolamine N-methyltransferase (PNMT) (C) and GABA_B-R (D) antibodies in the same section (A and B, C and D) of the rat adrenal medulla. GABA_B-R-immunoreactivity is seen in the ganglion cells (asterisks) in the medulla (B, D). No GABA_B-R-immunoreactivity is observed in noradrenaline cells (NA) demonstrating blue-white fluorescence and in adrenaline cells (A) demonstrating PNMT-immunoreactivity (A and B, C and D). Scale bar: 40 μm.

Figure 9

Fluorescent micrographs of double-immunostaining of gamma-aminobutyric acid_B-receptor (GABA_B-R) (A, C, E), and neuropeptide tyrosine (NPY) (B, D) and nitric oxide synthase (NOS) (F) antibodies in the same section (A and B, C and D, E and F) of the rat adrenal medulla. GABA_B-R-immunoreactive ganglion cells (A) (asterisks) are all NPY-immunopositive (B) (asterisks). GABA_B-R-immunoreactive ganglion cells (C) (asterisks) are all NPY-immunonegative (D) (arrows). GABA_B-R-immunoreactive ganglion cells (asterisks) are all NOS-immunopositive (F) (asterisks). Scale bar: 50 μm.

Figure 10

Fluorescent micrographs showing replacement of the primary antibody with normal rabbit serum in the sections of the rat adrenal medulla (A) and cortex (B). No immunoreactivity is seen in the cortex (A) and in the medulla (B). C, capsule; Co, cortex; M, medulla; V, blood vessel. Scale bar: 60 μm.