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

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.

γ-Aminobutyric acid B receptor immunoreactivity in the mouse adrenal medulla.

The present study examined gamma-aminobutyric acid B (GABAB ) receptor, GABA, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) immunoreactivities in the mouse adrenal medulla. GABAB receptor immunoreactivity was seen in numerous chromaffin cells and in a few ganglion cells of the adrenal medulla. By using a formaldehyde-induced fluorescence (FIF) method, GABAB receptor immunoreactivity was observed in numerous adrenaline (A) cells, but not in noradrenaline (NA) cells showing blue-white fluorescence. This suggests that GABAB receptors may be present in the A cells and be related to the secretory activity of A cells but not NA cells in the mouse adrenal medulla. GABAB receptor immunoreactive ganglion cells were shown to be nNOS immunopositive by using a double immunostaining method. Weak GABA immunoreactivity was visible in some chromaffin cells and in the numerous nerve fibers of the medulla. By using the FIF method, weak GABA-immunoreactive chromaffin cells were shown to be in the NA cells showing blue-white fluorescence. GABA-immunoreactive nerve fibers were in dense contact in A cells, but not NA cells. GABA-immunoreactive nerve fibers closely contacted a few ganglion cells. Numerous GABA-immunoreactive nerve fibers in the medulla showed ChAT immunoreactive. This result suggests that GABA and acetylcholine may be released from the same nerve fibers and may have a secretory effect on the A cells of the medulla.

Substance P immunoreactivity in the rat adrenal gland during postnatal development.

Substance P (SP)-immunoreactive nerve fibers in the adrenal medulla was very few during postnatal day 0-5, indicating that its synthesis in the neurons and the transport to nerve endings was incomplete by the end of this period. Since the number of SP-immunoreactive nerve fibers gradually increased during postnatal week 1-2, it was hypothesized that SP was not fully transported to nerve endings until postnatal week 1-2. At postnatal week 3, numerous SP-immunoreactive nerve fibers contacted some noradrenaline (NA) cells but not adrenaline (A) cells in the medulla. From postnatal week 3 onward, the abundance and expression patterns of SP-immunoreactive nerve fibers in the medulla were similar to those in adults. At postnatal week 3, the innervation with SP-immunoreactive nerve fibers was completed indicating the possibility that SP affected on the secretory activity of NA cells but not of A cells in the medulla. The medullary SP-immunoreactive nerve fibers contacting the chromaffin cells possessed a few dense-cored vesicles in their endings at postnatal week 8. Very few SP-immunoreactive chromaffin cells were found in the medulla from postnatal day 0 onward, and SP immunoreactivity was primarily observed in granular cores of the cells suggesting that SP and catecholamine synthesized in the chromaffin cells were released from the granules by adequate stimuli. Very few or a few SP-immunoreactive nerve fibers, acting as a vasomotor effect were found around blood vessels in the superficial cortex from postnatal day 0 onward.

Acetylcholinesterase activity, choline acetyltransferase and vesicular acetylcholine transporter immunoreactivities in the rat adrenal gland during postnatal development.

From postnatal-day-0 to postnatal-day-2, a few acetylcholinesterase (AChE)-active and choline acetytransferase (ChAT)-immunoreactive nerve fibers and relatively numerous vesicular acetylcholine transporter (VAChT)-immunoreactive puncta were observed in the rat adrenal medulla. Despite relatively numerous clear vesicles in the nerve fibers, the synthesis and hydrolysis of acetylcholine may not be fully activated until postnatal-day-2. The number of AChE-active and ChAT-immunoreactive nerve fibers dramatically increased and that of VAChT-immunoreactive puncta gradually increased from postnatal-day-3 to postnatal-week-1. The synthesis and hydrolysis of acetylcholine may be dramatically activated in the nerve fibers of the medulla until postnatal-week-1. From postnatal-week-2 to postnatal-week-3, the number of AChE-active and the ChAT-immunoreactive nerve fibers gradually increased and reached the adult levels. The VAChT-immunoreactive puncta per unit area was maximum number at postnatal-week-2. The synthesis and hydrolysis of acetylcholine in the nerve fibers of the medulla may be completed between postnatal-week-2 to postnatal-week-3. The diameter of the VAChT-immunoreactive puncta gradually increased from postnatal-day-0 with aging. However, the number of the VAChT-immunoreactive puncta gradually decreased from postnatal-week-2 onwards. In electron-microscopy, the VAChT-immunoreactive deposits were seen in clusters of clear vesicles, and the diameter of the nerve fibers and the number of clear vesicles at postnatal-week-8 increased compared with those at postnatal-week-2. The AChE-active, ChAT-immunoreactive, and VAChT-immunoreactive nerve fibers observed around noradrenaline (NA) cells were denser than those around adrenaline (A) cells in the medulla at postnatal-week-8. These suggest that the preferential innervation of NA and A cells may cause the differential secretion NA and A.

Immunohistochemical features of substance P-immunoreactive chromaffin cells and nerve fibers in the rat adrenal gland.

The distribution of substance P (SP) immunoreactivity and the colocalization of SP with other bioactive substances in chromaffin cells and nerve fibers were investigated in the rat adrenal gland at the light microscopic level. In the capsule and cortex, SP immunoreactivity was seen in some nerve fibers around blood vessels and in thick nerve bundles passing through the cortex directly into the medulla. In the medulla, the SP immunoreactivity was observed in a small number of chromaffin cells; these SP-immunoreactive chromaffin cells were either phenylethanolamine N-methyltransferase (PNMT) immunoreactive or immunonegative, indicating that they were either adrenaline cells or noradrenaline (NA) cells. SP-immunoreactive varicose nerve fibers were also found in the medulla and were in contact with a cluster of the NA cells showing catecholamine fluorescence, which suggests that SP from medullary nerve fibers may regulate the secretory activity of the NA cells. Because no SP-immunoreactive ganglion cell was present in the rat adrenal gland, the intra-adrenal nerve fibers were considered to be extrinsic in origin. The double-immunostaining method further revealed that the SP-immunoreactive chromaffin cells also exhibit immunoreactivities for calcitonin gene-related peptide (CGRP), and neuropeptide tyrosine (NPY), suggesting that these peptides can also be released from the chromaffin cells by certain stimuli. The intra-adrenal nerve fibers in the medulla were composed of SP-single immunoreactive, and SP/CGRP-, SP/choline acetyltransferase (ChAT)-, SP/nitric oxide synthase (NOS)-, SP/pituitary adenylate cyclase activating polypeptide (PACAP)-, ChAT/NOS-, and ChAT/PACAP-immunoreactive nerve fibers, which may affect the secretory activity of the NA cells. In the adrenal capsule, the nerve fibers were present around blood vessels and showed immunoreactivities for SP/ CGRP, SP/NPY, SP/NOS, and SP/vasoactive intestinal polypeptide, suggesting that the origin of nerve fibers in the capsule may differ from those in the medulla.

Localization of neurokinin 1 receptor (NK1R) immunoreactivity in rat esophagus.

The aim of the present immunohistochemical study was to investigate the localization of neurokinin 1 receptor (NK1R) in rat esophagus and examine the relationship between NK1Rs and intrinsic cholinergic, nitrergic, or substance P (SP) neurons. NK1R immunoreactivity (IR) was observed on the nerve cell bodies in the myenteric ganglia throughout the esophagus, but not on striated muscles and smooth muscle cells of the muscularis mucosae. The frequency of occurrence of NK1R neurons was highest in the cervical esophagus and lowest in the lower thoracic esophagus. Considerable immunoreactivity was seen on the nerve cell surfaces and was also present in the cytoplasm of cell somas and in the initial part of the axons, but not in any other nerve fibers or terminals. Dogiel type I-like morphology was observed in some of the NK1R neurons; however, the majority exhibited polymorphic morphology. Double immunolabeling indicated that a majority (77%) of the NK1R neurons were immunoreactive for choline acetyltransferase (ChAT), while a minority (23%) were immunoreactive for nitric oxide synthase (NOS)-IR. Most of the NK1R neurons (92%) were innervated by the SP nerve fibers. Triple immunolabeling indicated that 70% of the NK1R neurons were associated with intrinsic SP nerve fibers (without CGRP-IR), 59% were associated with extrinsic SP nerve fibers (with CGRP-IR), and 35% were associated with both intrinsic and extrinsic SP nerve fibers. These results suggest that SP/tachykinin released from the SP nerve fibers of intrinsic and/or extrinsic origin activates the predominantly intrinsic cholinergic neurons via NK1Rs to influence neuronal transmission or motility in rat esophagus.

Neuropeptide Y- and catecholamine-synthesizing enzymes: immunoreactivities in the rat carotid body during postnatal development.

Immunocytochemical studies of the rat carotid body during postnatal development revealed neuropeptide Y (NPY), tyrosine hydroxylase (TH), and dopamine beta-hydroxylase (DBH) immunoreactivities. In adult rats (at postnatal week 10), NPY and DBH immunoreactivities were shown in a few small chief cells (cell number/section shown as mean +/- SD: NPY 3.4+/-2.6, DBH 3.2+/-2.3), in large ganglion cells, and in numerous varicose nerve fibers of the carotid body. TH immunoreactivity was found in almost all chief cells, in a few ganglion cells, and in numerous varicose nerve fibers in the carotid body. By using the double-immunostaining technique, most NPY-immunoreactive chief cells, ganglion cells, and nerve fibers exhibited DBH immunoreactivity. The NPY- and DBH-immunoreactive chief cells in the rat carotid body were numerous from birth (NPY 93.8+/-14.9, DBH 89.7+/-12.3) to postnatal week 1 (NPY 65+/-14.5, DBH 61.6+/-11.3), but decreased quickly from postnatal week 2 (NPY 6.1+/-3.5, DBH 3.6+/-2.8) onwards. A few NPY- and DBH-immunoreactive ganglion cells were found in the periphery or in the center of the rat carotid body during postnatal development. TH immunoreactivity was observed in almost all chief cells and in a few ganglion cells in all developmental stages. NPY- and DBH-immunoreactive nerve fibers were very scarce in the carotid body from birth to postnatal week 1, began to increase gradually after postnatal week 2, and reached the adult level by postnatal week 5. The present study suggests that the expression of NPY and noradrenaline in chief cells and in the nerve fibers of the rat carotid body may be regulated during postnatal development.