Search

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.

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.

From postnatl-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. Anat Rec, 292:371–380, 2009. © 2009 Wiley-Liss, Inc.

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.

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.

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.

The present study showed the acetylcholinesterase (AChE) activity, and neurofilament protein (NFP), catecholamine-synthesizing enzymes, dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) immunoreactivities in the mouse adrenal gland during postnatal development. From birth to postnatal-1-day, AChE activity was weakly and diffusely found in some medullary cells and in very few nerve fibers whereas strong NFP immunoreactivity was seen in a few ganglion cells and in remarkably numerous nerve fibers in the medulla. Almost all meduallary cells were reactive for both DBH and PNMT during this period. From postnatal-2- or -3-day to postnatal-1-week, strong AChE activity was observed in a few large ganglion cells, but the reaction was weak in clusters of chromaffin cells, and the number of strong AChE-active nerve fibers in the medulla was rapidly increased. From postnatal-2-day onwards, the number of NFP-immunoreactive nerve fibers in the medulla were remarkably numerous. Numerous chromaffin cells were reactive for both DBH and PNMT whereas some chromaffin cells were reactive for only DBH from postnatal-2-day onwards. These results suggest that drastic changes such as an increase of acetylcholine in the nerve fibers, differentiation of noradrenaline and adrenaline cells of the medulla may occur during this period. From postnatal-2-week to postnatal-3-week, weak AChE activity was seen in the clusters of several chromaffin cells and a few ganglion cells, and the number of AChE-active nerve fibers in the medulla was gradually increased. From postnatal-4-week to postnatal-8-week (adult), the distribution and frequency of AChE activity in the adrenal gland were similar to those at postnatal-3-week. In the adult, AChE activity was weakly seen in the clusters of several chromaffin cells showing noradrenaline fluorescence in the adrenal medulla. The noradrenaline cells were contacted by denser AChE-reactive nerve fibers than adrenaline cells. These results suggest that the development of cholinergic nervous system in the mouse adrenal medulla may be completed by postnatal-3-week.

The present immunohistochemical and enzyme histochemical study showed gamma-aminobutyric acid (GABA) immunoreactivity and acetylcholinesterase (AChE) activity in the mouse adrenal gland. Weak GABA immunoreactivity was seen in clusters of chromaffin cells showing noradrenaline fluorescence. This finding suggests that both GABA and noradrenaline may be released from the granules of noradrenaline cells by adequate stimuli. GABA-immunoreactive varicose nerve fibers densely contacted adrenaline cells and large ganglion cells, but they were sparse in the periphery of clusters of noradrenaline cells. AChE activity was strong in a few large ganglion cells and weak in chromaffin cells showing noradrenaline fluorescence, and was found in numerous nerve bundles and fibers of the medulla. AChE-active nerve fibers more densely contacted noradrenaline cells than adrenaline cells. By using double labeling technique, numerous GABA-immunoreactive nerve fibers in the medulla were reactive for AChE in the same sections. These results suggest that both GABA and acetylcholine may be colocalized in the intra-adrenal nerve fibers and may have some secretory effects on the chromaffin cells.

Monoclonal antibody (MAb) BEG3 was produced against Babesia equi parasites to define a species-specific antigen for diagnostic use. The MAb reacted with single, paired, and Maltese cross forms of B. equi , and no reaction was observed with this MAb on acetone-fixed Babesia caballi , Babesia ovata , or Babesia microti parasites in the indirect immunofluorescent antibody test. Confocal laser and immunoelectron microscopic studies showed that the antigen which was recognized by this MAb was located on the surface of B. equi parasites. This MAb recognized a 19-kDa protein of B. equi antigen and did not react with B. caballi antigen or normal horse erythrocytes in immunoblot analysis. This MAb also significantly inhibited the in vitro growth of the B. equi parasite. Preliminary studies using partially purified antigen, which was separated by high-pressure liquid chromatography and recognized by the MAb, suggested that it is a suitable antigen for enzyme-linked immunosorbent assay detection of anti- B. equi antibodies in naturally infected horse sera.

Langerhans cell histiocytosis is currently regarded as a reactive proliferative process of Langerhans cells rather than a malignancy. The disease is characterized by Langerhans cell infiltration of skin, lung, bone and other organs. We report a 74-year-old man with Langerhans cell histiocytosis who had generalized hemorrhagic and crusted papules. He also had diabetes insipidus. Because he did not have any severe constitutional symptoms or failure of vital organs, we applied topical PUVA treatment to his skin lesions, which responded well to the therapy. Diabetes insipidus, however, remained, in spite of X ray radiotherapy for the pituitary lesion.

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.

The immunohistochemical study revealed tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), serotonin, glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) immunoreactivities in the mouse carotid body. TH and DBH immunoreactivities were found in almost all chief cells and a few ganglion cells, and in relatively numerous varicose nerve fibers of the carotid body. The histofluorescence microscopy showed catecholamine fluorescence in almost all chief cells. However, no PNMT immunoreactivity was observed in the carotid body. Serotonin, GAD and GABA immunoreactivities were also seen in almost all chief cells of the carotid body. From combined immunohistochemistry and fluorescence histochemistry, catecholamine and serotonin or catecholamine and GABA were colocalized in almost all chief cells. Thus, these findings suggest that noradrenaline, serotonin and GABA may be synthesized and co-exist in almost all chief cells of the mouse carotid body and may play roles in chemoreceptive functions.

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intra-adrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.

Gamma-aminobutyric acid (GABA) immunoreactivity was revealed by immunocytochemistry in the mouse adrenal gland at the light and electron microscopic levels. Groups of weakly or faintly GABA immunoreactive chromaffin cells were often seen in the adrenal medulla. By means of immunohistochemistry combined with fluorescent microscopy, these GABA immunoreactive chromaffin cells showed noradrenaline fluorescence. The immunoreaction product was seen mainly in the granular cores of these noradrenaline cells. These results suggest the co-existence of GABA and noradrenaline within the chromaffin granules. Sometimes thick or thin bundles of GABA immunoreactive nerve fibers with or without varicosities were found running through the cortex directly into the medulla. In the medulla, GABA immunoreactive varicose nerve fibers were numerous and were often in close contact with small adrenaline cells and large ganglion cells; a few, however, surrounded clusters of the noradrenaline cells, where membrane specializations were formed. Single GABA immunoreactive nerve fibers, and thin or thick bundles of the immunoreactive varicose nerve fibers ran along the blood vessels in the medulla. The immunoreaction deposits were observed diffusely in the axoplasm and in small agranular vesicles of the GABA immunoreactive nerve fibers. Since no ganglion cells with GABA immunoreactivity were found in the adrenal gland, the GABA immunoreactive nerve fibers are regarded as extrinsic in origin.

The immunolight- and electron-microscopic study revealed neuropeptide Y (NPY) immunoreactivity in the chief cells and a few nerve fibers of the carotid body of young rats. NP Y-immunoreactive chief cells were often seen as a single cell or a group of a few chief cells in the carotid body of young rats. The immunoreaction deposits were mainly seen in the dense-cored vesicles and diffusely in the cytoplasm of the chief cells. These findings suggest that NPY may be stored in the core of granules of the chief cells of the carotid body in young rats.

An electron-microscopic study revealed exocytosis of electron-dense granules in rat pheochromocytoma (PC12) cells after 1-min exposure to a high concentration of potassium medium. Numerous omega profiles indicative of the exocytosis were seen in the surface membrane of the PC12 cells. Most omega profiles showed clear or did not contain the granule contents. Sometimes, the granular, flocculent or dense materials were seen within the omega profiles that were exposed directly to the extracellular medium. Moreover, noncoated or coated omega profiles were observed. Coated vesicles were also visible under the surface membrane. The present study suggests that PC12 cells retain the exocytotic release mechanism for catecholamines.

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.

To understand the developing processes of gamma-aminobutyric acid (GABA)-containing chromaffin cells and nerve fibers in the mouse adrenal gland, we examined the tissues in various postnatal stages by immunohistochemistry using a GABA antibody. From birth until postnatal week 1, GABA-immunoreactivity was seen in very few nerve fibers, and in none of the chromaffin cells. At postnatal week 2, GABA-immunoreactivity appeared weakly in clusters of chromaffin cells and strongly in relatively numerous varicose nerve fibers. The immunoreactive nerve fibers were densely distributed in the small immunonegative chromaffin cells and large ganglion cells, but only sparsely so in the weak immunoreactive chromaffin cells. At postnatal week 3, the number of the immunoreactive chromaffin cells and nerve fibers further increased compared to that at postnatal week 2. The staining pattern of GABA-immunoreactive nerve fibers in the medullas was similar to that at postnatal week 2. From postnatal week 4 until postnatal week 8, the distribution and frequency of the immunoreactive chromaffin cells and nerve fibers were also similar to those at postnatal week 3. These results suggest that the expression of GABA in the chromaffin cells and in the nerve fibers of the mouse adrenal gland may be completed by postnatal week 3.

To clarify when the cholinergic receptor-mediated secretion mechanism of developing adrenal chromaffin cells is expressed and becomes functional, morphological changes and intracellular calcium dynamics were studied by immunohistochemistry, electron microscopy, and Fura-2 digital image analysis. From embryonic day 14 to 16, adrenal medullary cells were immunoreactive to noradrenaline-synthesizing enzyme (dopamine beta-hydroxylase) but not to adrenaline-synthesizing enzyme (phenylethanolamine N-methyltransferase). These cells contained either no granules or just a few granules of high electron density. Exocytotic figures were rarely observed in cells of the control or in cells after carbamylcholine stimulation. Nerve fibers in the adrenal medulla contained either no clear vesicles or very few. Neither methacholine nor nicotine caused a change of intracellular Ca2+ in most chromaffin cells. From embryonic day 18 to 20, chromaffin cells were immunoreactive to both dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase and they contained relatively numerous secretory granules. Exocytotic figures were often seen in cells after carbamylcholine stimulation. The intra-adrenal nerve fibers contained numerous clear vesicles and a few dense-cored vesicles. Methacholine caused no rise of intracellular Ca2+, but nicotine induced a low to relatively high rise in many cells. From postnatal day 2 or 3 to postnatal week 1, numerous cells were immunoreactive to both dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase, whereas some cells were reactive to dopamine beta-hydroxylase alone. Chromaffin cells were divisible into noradrenaline cells and adrenaline cells based on the ultrastructural features of their granules. Methacholine induced a moderate rise of intracellular Ca2+ and nicotine caused a high rise in many chromaffin cells, whereas, in some chromaffin cells, methacholine induced no rise of intracellular Ca2+ and nicotine induced a high rise. These results suggest that morphological changes of the developing cells and the intra-adrenal nerve fibers are related to the expression of a cholinergic receptor-mediated secretion mechanism and that this mechanism via a nicotinic receptor-mediated Ca2+ signaling pathway precedes the muscarinic receptor-mediated one during development.

In order to capture the exocytotic figures of gastrin cells in the pyloric antrum of the stomach, we examined antral cells of the mouse, rat, and guinea pig by electron microscopy following stimulation with the cholinergic secretagogue carbamylcholine. Increased numbers of omega profiles indicative of exocytosis were seen in the basal or lateral cell membrane after stimulation with carbamylcholine. The number of exocytotic figures in stimulated gastrin cells was higher in the guinea pig than in the mouse and rat. Coated and non-coated omega profiles and coated pits in the plasma membrane were smaller than the secretory granules. Omega profiles with or without electron-dense contents were seen. Coated and non-coated vesicles were often visible near the plasma membrane of stimulated gastrin cells in all three species, large cytoplasmic vacuoles also being found in the guinea pig. In the mouse pretreated with horseradish peroxidase, reaction deposits were observed in the omega profiles and in microvesicles near the plasma membrane. These results suggest that, after exocytosis, membrane retrieval and endocytosis occur in the gastrin cells.

The present study shows gamma-aminobutyric acid (GABA) immunoreactivity in almost all chief cells of the mouse carotid body. GABA immunoreaction products were distributed diffusely in the cytoplasm as well as in the dense-cored granules. These findings suggest that GABA may be localized in the cytoplasm and granules, and may be released from the cells.

An immunohistochemical and immunoelectron microscopic study was used to demonstrate tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) immunoreactivities in the rat pancreas. Small TH immunoreactive cells were found in close contact with large TH immunonegative ganglion cells among the exocrine glands and were occasionally found in some islets. Some of these TH immunoreactive cells were also DBH immunopositive. The immunoreaction product was seen diffusely in the cytoplasm and in the granule cores of TH immunoreactive cells. All intra-pancreatic ganglion cells were immunoreactive for DBH, but not for TH. The TH immunoreactive cells were identified as small intensely fluorescent (SIF) cells due to their localization and morphological characteristics and showed no insulin, glucagon, somatostatin or pancreatic polypeptide immunoreactivities. These results indicate that SIF cells may release dopamine or noradrenaline to adequate stimuli while the intra-pancreatic ganglion cells with only DBH may not synthesize catecholamines in a normal biosynthetic pathway. TH immunoreactive nerve bundles without varicosities and fibers with varicosities, associated or unassociated with blood vessels, were found in both the exocrine and endocrine pancreas. Close apposition of TH immunoreactive nerve fibers to the smooth muscle and endothelial cells of the blood vessels was observed. A close apposition between TH immunoreactive nerve fibers and exocrine acinar cells and islet endocrine cells was sometimes found in the pancreas. The immunoreaction product was seen diffusely in the axoplasm and in the granular vesicles of the immunoreactive nerve fibers. Since no TH immunoreactive ganglion cells were present in the rat pancreas, the present study suggests that noradrenergic nerve fibers in the pancreas may be extrinsic in origin, and may exert an effect on the regulation of blood flow and on the secretory activity of the acinar cells, duct cells and endocrine cells.

To study the configuration of myoepithelial cells, we isolated glandular endpieces of various guinea pig glands by collagenase, and visualized the myoepithelial cells by immunohistochemistry for actin, or by Bodipy-phallacidin, under both a light microscope and laser scanning confocal microscopes. In parotid and mandibular glands, the glandular acini were small (about 20-30 microns diameter) and spherical, and each acinus had one or two myoepithelial cells attached that were stellate in shape (central cell body and four to six thin processes). Most of the basal surface of the glandular cells was not covered by myoepithelial cells, and processes often extended to the neighboring acinus. The tubular glandular endpieces of the major sublingual gland, which secretes a mucous substance, were almost fully encircled by band-like myoepithelial cells (about 3-6 microns wide). Although there were many differences between the lacrimal gland and the Harderian gland (e.g., the secretory product of the lacrimal gland was mucous, and glandular lumina were narrow; the Harderian gland secreted lipids and showed wide lumina), the outer contours of both glandular endpieces were the same (about 50-100 microns diameter, ellipsoid or spherical in shape). In both glands, 5-20 stellate myoepithelial cells were attached onto a glandular endpiece, and their arrangement had a lacy appearance. Actin filaments in myoepithelial cells aggregated and formed bundles in the broad processes and cell bodies. The bundles ran across the cell body, but there was no point where the bundles converged. In the arborization, some distal processes reversed their direction. We conclude that the configuration of myoepithelial cells depends on the outer contour of the glandular endpieces rather than on the secretory material or luminal width. The variety of myoepithelial cell configurations in the different exocrine glands we examined suggests that it is quite difficult to assign to myoepithelial cells the general role of expelling secretory products from glandular lumina. These cells seem to maintain the contour of the glandular endpieces, serving as the exoskeleton of the endpieces.

Summary After fixation at pH = 7 or pH = 5 the gastrin ( G ) cells in the rat pyloric antrum were investigated by conventional and immunoelectron microscopy. After fixation at pH = 7 G cells contained numerous electron-lucent granules, a few electron-dense and intermediate granules while G cells had numerous electron-dense and intermediate granules, and a few electron-lucent granules after fixation at pH = 5. Preembedding immunoelectron microscopy revealed that in G cells after fixation at pH = 7, gastrin immunoreactivity was mainly seen in the cytoplasm, cores of a few electron- dense and intermediate granules and in the periphery but not within the numerous electron-lucent granules; in G cells after fixation at pH = 5, gastrin immunoreactivity was mainly visible in the cores of numerous intermediate and electron-dense granules, in the periphery of a few electron-lucent granules, but weakly in the cytoplasm. Therefore, the variable electron density of G cells may reflect differences in the degree of the leakage of contents from the gastrum-containing granules into the cytoplasm during fixation. This phenomenon may be related to the acidity of the granule substances during intragranular maturation.

Paneth cells located at the bottom of intestinal crypts may play a role in controlling the bacterial milieu of the intestine. Using morphometry to clarify the secretory mechanism of the Paneth cells, we studied the ultrastructural changes in mouse Paneth cells produced following intra-arterial perfusion with Hanks' balanced salt solution containing a cholinergic muscarinic secretagogue (bethanechol), a neuroblocking agent (tetrodotoxin), or a G-protein activator (NAF/AlCl3). Bethanechol (2 x 10(-4) mol/l) induced Paneth-cell secretion. Many Paneth cells massively exocytosed their secretory material into the crypt lumen; the enhanced secretion caused degranulation and vacuole formation. However, tetrodotoxin (2 x 10(-6) mol/l) did not prevent the bethanechol-enhanced secretion by the Paneth cells. NaF (1 x 10(-2) mol/l) and AlCl3 (1 x 10(-5) mol/l) induced massive exocytosis of the Paneth cells; the exocytotic figures were similar to those observed in mice stimulated by bethanechol. G-protein activation was followed by a sequence of intracellular events, resulting in exocytosis.

The present immunocytochemical study revealed γ-aminobutyric acid (GABA) immunoreactivity in the oxyntic and pyloric mucosa of the rat stomach at light-and electron-microscopic levels. GABA-immunoreactive endocrine cells were numerously seen in the lower half portion of the pyloric mucosa but rarely in the oxyntic mucosa. These cells were round or oval in shape and sometimes had a short cytoplasmic process. Serotonin-immunoreactive enterochromaffin (EC) cells were also observed in the oxyntic and pyloric mucosa of the stomach. The distribution and shapes of the immunoreactive cells were similar to those of the GABA-immunoreactive cells. With a double immunolabeling technique using anti-GABA and antiserotonin serum, GABA-immunoreactive endocrine cells showed serotonin immunoreactivity and were identified as EC cells. At the electron-microscopic level the GABA-immunoreactive cells contained round or oval, spindle-like, pear-shaped granules in EC cells. The immunoreaction product in the EC cells was generally confined to the granular cores. These findings suggest that GAB A may be synthesized in the EC cells and be released from the granules of the cells after adequate stimuli.

The present immunocytochemical study used an antiserum to tyrosine hydroxylase (TH), the first enzyme in the biosynthetic pathway of catecholamines, and revealed TH immunoreactivity in the ganglion cells and in the varicose nerve fibers of the cortex and medulla in the rat adrenal gland. TH immunoreactive nerve fibers in the cortex and medulla contained large and small granular vesicles, and also small clear vesicles. The immunoreactive nerve fibers were in close apposition to cortical cells in the cortex and in apposition to smooth muscle cells of blood vessels in both the cortex and medulla. Furthermore, TH immunoreactive nerve fibers were sometimes in close apposition to pericytes of blood vessels in the cortex and chromaffin cells in the medulla. The present results suggest that the catecholaminergic nerve fibers in the rat adrenal gland may be both intrinsic and extrinsic in origin.

The ultrastructure of annulate lamellae in the pancreatic exocrine cells of rats starved for 3-42 days was studied. Annulate lamellae were rarely encountered in the pancreatic exocrine cells of control rats, but their incidence and size of stack was increased in those of starved rats depending on the duration of starvation, despite marked ultrastructural changes in these cells. Annulate lamellae were located in the para- or infranuclear cytoplasm of the cells and most of them were continuous with the relative normal rough endoplasmic reticulum. A close apposition in parallel was often found between the annulate lamellae and the outer nuclear envelope and occasionally pores in line with those of the nuclear membrane were observed in the lamellae of rough endoplasmic reticulum in 35- and 42-day-starved rats. An unequivocal continuity between the annulate lamellae and the nucleus and/or rough endoplasmic reticulum in pancreatic exocrine cells might be related to their origin and function.

The gastroenteropancreatic (GEP) endocrine cells of the sheep were studied immunocytochemically and their distribution and frequency were determined. Eleven types of endocrine cells were revealed. In the abomasum, somatostatin-, gastrin-, glucagon- and glicentin-immunoreactive cells were detected with the highest frequency in the pyloric region. In the small intestine, somatostatin-, gastrin-, CCK-, motilin-, neurotensin-, secretin-, substance P-, glucagon-, glicentin- and BPP-immunoreactive cells were found and were most numerous in the duodenum except for neurotensin-, glucagon- and glicentin-immunoreactive cells which were more concentrated in the ileum. In the large intestine, somatostatin-, substance P-, glucagon-, glicentin- and BPP-immunoreactive cells were localized with the last three cell types being more concentrated in the rectum. In the pancreas, somatostatin-, glucagon-, glicentin-, BPP- and insulin-immunoreactive cells predominated within the islets and were also scattered in the exocrine portion and rarely detected in duct epithelial cells. The differences between the distribution and frequency of the GEP endocrine cells of the sheep and those of monogastric species are discussed.

The present study applied the separated adrenal capsules of rats for wholemount immunocytochemistry and used tyrosine hydroxylase (TH) antibody as a marker for catecholamines. TH-immunoreactive nerve bundles without varicosities and fibers with varicosities were seen to run along or to encircle blood vessels entering the adrenal capsule from the outside, and then to run along a network of blood vessels in the intracapsular region. Also, the TH-immunoreactive nerve bundles and fibers were found to run along blood vessels in the subcapsular region. Some TH-immunoreactive nerve fibers and bundles with varicosities unassociated with the blood vessels were seen in the subcapsular region. In this region, TH-immunoreactive nerve fibers with varicosities were often seen to be closely associated with the cortical cells. Some TH-immunoreactive nerve fibers without varicosities were visible within the splanchnic nerve in the subcapsular region. The present study suggests that numerous catecholaminergic nerve fibers are associated with blood vessels forming a network in the superficial region of the rat adrenal gland.

Summary Ultrastructural localization of thiamine pyrophosphatase (TPPase) activity was studied in the small and large intestinal epithelial cells of adult rats. In the duodenal and jejunal absorptive cells, TPPase activity was localized on the microvilli, on the lateral cell membrane, and on the lysosome-like bodies. In the ileal epithelial cells, TPPase activity was detected on the lysosome-like bodies, and weak phosphatase activity was observed on the microvilli and on the cisternae of the rough-surfaced endoplasmic reticulum (rER). In the principal cells of the caecum, the ascending colon, and the descending colon, the lysosome-like bodies and the cisternae of the rER showed TPPase activity. Almost all Golgi lamellae, vesicles and GERL elements ( Novikoff 1964) in the duodenal absorptive cells were strongly TPPase positive. The 1st to 3rd of the inner Golgi lamellae and the vesicles of the jejunal, ileal, and large intestinal epithelial cells usually showed positive TPPase activity, while the GERL elements were totally negative.

The structural changes of the cecal wall in germ-free rats were observed at regular intervals after the inoculation of fecal microflora from conventional rats. Quantitative light microscopy showed that most of the elements in the cecal wall increased at 12 or 24 h and reached peak values at 4 days after inoculation. On the 7th day, they decreased approximately to the values for conventional rats. The crypts were bent or widely open till 24 h but were not after the 4th day. Hyperplasia of the crypt epithelial cells including mucous-type cells was observed following microbial inoculation. Electron microscopy revealed that most of the epithelial cells lining the mucosa were typical columnar cells. Desquamation of the epithelial cells and contraction of the muscle fibers were often seen on 4th day. The mucous-type cells were divided into two types, goblet and non-goblet mucous-type cells. Reduction of cecal volume after microbial inoculation may be mainly caused by muscle contraction in the early period and hyperplasia and desquamation of the epithelial cells may suggest their role as the first and non-specific defense line prior to operation of the specific immune system.

Summary The ultrastructure of annulate lamellae of the jejunal absorptive cells in control and 21 d starved rats was investigated. Annulate lamellae were only rarely encountered in the jejunal absorptive cells of control rats, and then frequently in small stacks continuous with the rough-surfaced endoplasmic reticulum. In contrast, there was a relatively frequent incidence of annulate lamellae in the jejunal absorptive cells of 21 d starved rats, and larger stacks of annulate lamellae were also observed in spite of marked ultrastructural changes of these cells. The annulate lamellae were also continuous with the rough-surfaced endoplasmic reticulum, which was degenerating. The degenerative process of the absorptive cells following starvation might be related to the origin and function of the annulate lamellae.

To confirm whether the Paneth cells of mice (ICR, male, 10-12 weeks old) have the same secretory response to hormonal and cholinergic stimulation as do pancreatic acinar cells, ultrastructural changes of Paneth cells and pancreatic acinar cells 1 hr after administration of various doses of cholecystokinin (octapeptide, CCK-8) and carbamylcholine were morphometrically assessed. After maximal (1.5 micrograms/kg intraperitoneally [i.p.]) and supramaximal (15 micrograms/kg, i.p.) stimulation by CCK-8, pancreatic acinar cells showed, respectively, degranulation or disturbance of secretion (e.g., an increase in lysosome-like bodies, aggregation of zymogen granules). The Paneth cells, however, were almost unchanged in the parameters examined. After carbamylcholine injection (1,000 micrograms/kg, subcutaneously [s.c.]), both pancreatic acinar cells and Paneth cells showed degranulation. Paneth cells sometimes developed large vacuoles, probably formed after massive exocytosis; such vacuoles were not observed in pancreatic acinar cells. It is suggested that Paneth cells and pancreatic acinar cells have different secretory responses. Paneth cell secretion, which possibly plays a role in controlling the intestinal bacterial milieu, may be stimulated by cholinergic rather than hormonal mechanisms.

The gastrin cells (G cells) in the rat pyloric antrum after 7, 14, 21 and 28 days of starvation were investigated by immunohistochemistry and electron microscopy. In the peroxidase anti-peroxidase method for light microscopy, gastrin immunoreactive cells during starvation markedly decreased in number and size. Quantitative electron microscopy revealed that during starvation the number of electron-lucent granules were greatly reduced, but the number of electron-dense granules increased; the number of intermediate granules were not remarkably changed in G cells. These results may suggest that the synthesis of gastrin and granule maturation were greatly inhibited during long-term starvation.

Summary The localization of lysozyme, which may function as an antibacterial agent, was immunohistochemically studied on the mouse Paneth cell secretory granules showing bipartite substructures (central core and peripheral halo).The lysozyme immunoreactivity was observed in the core, but not in the halo.Even in germ-free mice, Paneth cells have many secretory granules and their cores show lysozyme immunoreactivity.It seems likely that mice Paneth cells possess the ability to produce secretory granules containing lysozyme in disregard of the intestinal bacterial milieu.

In the present study, in order to easily grasp whole images of somatostatin (D) cells, the isolated mucosa of the mouse gastrointestinal tract was immunohistochemically treated. The present study revealed that: (1) in the stomach, small-intestinal villi and colon, about 20% of the D cells extrude basal cytoplasmic processes, showing terminal expansions in many cases; on the other hand, in the crypts of the small intestine, few D cells possess basal processes, and (2) in the stomach, there is no determined tendency in the direction of the basal processes of the D cells; on the other hand, in the small-intestinal villi and colon, most D cell basal processes run toward the villus base and colon crypt bottoms. The direction of the basal processes of the D cells in the gastrointestinal tract seems to be mostly in favor of the migration pattern of epithelial cells described previously. It is likely that, if the targets of the D cells are near the D cells the basal process is not necessary for local secretion of somatostatin. During migration however, D cells might extrude basal processes to keep relationships with their targets.

The localization of tyrosine hydroxylase (TH) immunoreactivity in rat adrenal chromaffin and pheochromocytoma (PC12) cells was investigated by immunoelectron microscopy using monoclonal and polyclonal anti-sera against TH purified from rat adrenal medulla. Strong TH immunoreactivity was found uniformly in the granules of the adrenaline cells; the immunoreactivity was visible mainly within the periphery, but not in the clear space of the granules of the noradrenaline cells. In the PC12 cells, strongTH immunoreactivity was also observed uniformly in the granules. In addition, TH immunoreactivity was seen in the cytoplasm, the ribosomes attached to the endoplasmic reticulum and the free ribosomes of both the rat adrenal chromaffin and PC12 cells. These results suggest thatTH may be localized in the granules, cytoplasm and ribosomes of rat adrenal chromaffin and PC12 cells.