Substance P-like immunoreactivity in adrenal chromaffin cells and intra-adrenal nerve fibers of rats

The present peroxidase-antiperoxidase immunohistochemical study demonstrated a relatively small number of cells with substance P(SP)-like immunoreactivity in the adrenal medulla of rats. These cells were found alone or in small groups, were polygonal in shape and lacked long cytoplasmic processes. At immunoelectron microscopy, the immunoreactive cells were characterized by abundant granular vesicles, and the immunoreactive material was confined to the round core of the vesicles. Thus, it is suggested that SP co-exists with catecholamines in a population of chromaffin cells of the rat adrenal medulla. In addition a few SP-immunoreactive nerve fibers with varicosities were found in the adrenal medulla of rats. They extended between small clusters of chromaffin cells and had their dot-like terminals around and within the cell clusters. The SP-immunoreactive nerve fibers were characterized by the presence of abundant small clear vesicles mixed with a few large granular vesicles; the immunoreactivity appeared in the latter, but was also perfused throughout the entire axoplasm. The nerve fibers formed synapses on nonimmunoreactive chromaffin cells. Judging from the presence of bundles of SP-immunoreactive nerve fibers penetrating the adrenal capsule and cortex as well as the absence of SP-immunoreactive ganglion cells in the medulla, the intramedullary SP-immunoreactive nerve fibers seem to be extrinsic in origin.     

Calcitonin gene-related peptide (CGRP)-like immunoreactivity in scattered chromaffin cells and nerve fibers in the adrenal gland of rats

Summary The present immunohistochemical study reveals that a small number of chromaffin cells in the rat adrenal medulla exhibit CGRP-like immunoreactivity. All CGRP-immunoreactive cells were found to be chromaffin cells without noradrenaline fluorescence; from combined immunohistochemistry and fluorescence histochemistry we suggest that these are adrenaline cells. In addition, all CGRP-immunoreactive cells simultaneously exhibited NPY-like immunoreactivity. CGRP-chromaffin cells were characterized by abundant chromaffin granules with round cores in which the immunoreactive material was densely localized. These findings suggest the co-existence of CGRP, NPY and adrenaline within the chromaffin granules in a substantial number of chromaffin cells.Thicker and thinner nerve bundles, which included CGRP-immunoreactive nerve fibers, with or without varicosities, penetrated the adrenal capsule. Most of them passed through the cortex and entered the medulla directly, whereas others were distributed in subcapsular regions and among the cortical cells of the zona glomerulosa. Here the CGRP-fibers were in close contact with cortical cells. A few of the fibers supplying the cortex extended further into the medulla. The CGRP-immunoreactive fibers in the medulla were traced among and within small clusters of chromaffin cells and around ganglion cells. The CGRP-fibers were directly apposed to both CGRP-positive and negative chromaffin cells, as well as to ganglion cells. Immunoreactive fibers, which could not be found close to blood vessels, were characterized by the presence of numerous small clear vesicles mixed with a few large granular vesicles. The immunoreactive material was localized in the large granular vesicles and also in the axoplasm. Since no ganglion cells with CGRP-like immunoreactivity were found in the adrenal gland, the CGRP-fibers are regarded as extrinsic in origin. In double-immunofluorescence staining for CGRP and SP, all the SP-immunoreactive fibers corresponded to CGRP-immunoreactive ones in the adrenal gland. This suggests that CGRP-positive fibers in the adrenal gland may be derived from the spinal ganglia, as has been demonstrated with regard to the SP-nerve fibers.     

An immuno-electron-microscopic study of the localization of VIP-like immunoreactivity in the adrenal gland of the rat

Summary VIP-like immunoreactivity was revealed in a few chromaffin cells, medullary ganglion cells and a plexus of varicose nerve fibers in the superficial cortex and single varicose fibers in the juxtamedullary cortex and the medulla of the rat adrenal gland. VIP-like immunoreactive chromaffin cells were polygonal in shape without any distinct cytoplasmic processes and they appeared solitarily. Their cytoplasm contained abundant granular vesicles having a round core and the immunoreactive material was localized to the granular core. VIP-immunoreactive ganglion cells were multipolar and had large intracytoplasmic vacuoles. The immunoreactive material was localized not only in a few granular vesicles but also diffusely throughout the axoplasm. VIP-immunoreactive varicose nerve fibers in the superficial cortex were characterized by abundant small clear vesicles and some large granular vesicles, while those in the juxtamedullary cortex and medulla and the ganglionic processes were characterized by abundant large clear vesicles, as well as the same vesicular elements as contained in the nerves in the superficial cortex. The immunoreactive material was localized on the granular cores and diffusely in the axoplasm in both nerves. Based on the similarity and difference in the composition of the vesicles contained in individual nerves, it is likely that the VIP-immunoreactive nerve fibers in the medulla and the juxtamedullary cortex are derived from the medullary VIP-ganglion cells, while those in the superficial cortex are of extrinsic origin. The immunoreactive nerve fibers in both the cortex and the medulla were often in direct contact with cortical cells and chromaffin cells, where no membrane specializations were formed. The immunoreactive nerve fibers were sometimes associated with the smooth muscle cells and pericytes of small blood vessels in the superficial cortex. In addition they were often seen in close apposition to the fenestrated endothelial cells in the cortex and the medulla, only a common basal lamina intervening. Several possible mechanisms by which VIP may exert its effect in the adrenal gland are discussed.     

Gamma-aminobutyric acid (GABA) immunoreactivity in the mouse adrenal gland

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.     

Heterogeneous distribution of neurocalcin-immunoreactive nerve terminals in the mouse adrenal medulla

Neurocalcin is a novel calcium-binding protein found in bovine brain tissue. We investigated immunoreactivity for neurocalcin in the mouse adrenal medulla using light and electron microscopy. The immunoreactivity was present in nerve fibers, nerve terminals, and ganglion cells in the adrenal medulla, but chromaffin cells, sustentacular cells, and Schwann cells were negative in reaction. Nerve bundles containing neurocalcin-immunoreactive fibers passed through the adrenal cortex and extended into the medulla. Immunopositive nerve fibers branched off and projected varicose terminals around the chromaffin cells. These varicose terminals contained small and large-cored vesicles and made synapses with the chromaffin cells. We performed paraformaldehyde-induced fluorescence-histochemical studies for catecholamine combined with immunohistochemical studies for neurocalcin. Neurocalcin-immunoreactive nerve terminals were more abundant at noradrenaline (fluorescent) cell-rich regions than at adrenaline (non-fluorescent) cell-rich regions. These results show that neurocalcin-immunoreactive nerves mainly innervate noradrenaline-containing chromaffin cells in the mouse adrenal medulla and that neurocalcin may regulate synaptic function in the nerve terminals. Received: 21 October 1996 / Accepted: 12 February 1997     

Somatostatin immunoreactivity in the cat adrenal medulla. Localization and characterization

Somatostatin-like immunoreactivity was detected within the adrenal gland of the cat using specific monoclonal antibodies. Immunohistochemical studies demonstrated a few somatostatin-immunoreactive nerve fibers within the adrenal medulla. In addition, a large population of chromaffin cells in the cat adrenal medulla displayed intense somatostatin-like immunoreactivity. Similar cells were not observed in rat or guinea pig adrenal glands, although they were found in human material. The somatostatin-positive cells in the cat adrenal medulla often possessed short immunoreactive processes similar to those seen in somatostatin-immunoreactive paracrine cells of the gut. Characterization of the somatostatin-like immunoreactivity of the cat adrenal by high performance liquid chromatography and radioimmunoassay indicated that somatostatin-28 may account for over 90% of the observed immunoreactivity. It is suggested that somatostatin-28 may have a paracrine or endocrine role in the feline adrenal medulla.     

Somatostatin immunoreactivity in the cat adrenal medulla

Summary Somatostatin-like immunoreactivity was detected within the adrenal gland of the rat using specific monoclonal antibodies. Immunohistochemical studies demonstrated a few somatostatin-immunoreactive nerve fibers within the adrenal medulla. In addition, a large population of chromaffin cells in the cat adrenal medulla displayed intense somatostatin-like immunoreactivity. Similar cells were not observed in rat or guinea pig adrenal glands, although they were found in human material. The somatostatin-positive cells in the cat adrenal medulla often possessed short immunoreactive processes similar to those seen in somatostatin-immunoreactive paracrine cells of the gut. Characterization of the somatostatin-like immunoreactivity of the cat adrenal by high performance liquid chromatography and radioimmunoassay indicated that somatostatin-28 may account for over 90% of the observed immunoreactivity. It is suggested that somatostatin-28 may have a paracrine or endocrine role in the feline adrenal medulla.     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--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 intraadrenal 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.     

The distribution and origin of nerve fibers immunoreactive for substance P and neurokinin A in the anterior buccal gland of the rat

The distribution and origin of substance P (SP) and neurokinin A (NKA) were studied in rat in the anterior buccal glands, which are minor mucous salivary glands. Indirect immunofluorescence staining showed moderate SP and NKA innervation of salivary acini and interlobular ducts, whereas blood vessels were more sparsely innervated, and there were few nerve fibers in the stroma and around the intralobular ducts. About 10%–20% of the trigeminal ganglion cells showed equally strong immunoreactivity to both SP and NKA. Unilateral denervation of the branches of the trigeminal nerve caused complete disappearance of the stromal fibers and greatly reduced the number of all other SP-immunoreactive and NKA-immunoreactive nerve fibers. In the superior cervical ganglia, SP and NKA immunoreactivity was restricted to small intensely fluorescent cells; SP and NKA immunoreactivity was absent from principal ganglionic cells, and thus sympathectomy had no any effect on the number or distribution of fibers immunoreactive for SP and NKA in the anterior buccal glands. The fibers remaining after sensory denervation could have been of parasympathetic origin, indicating a dual origin of nerves immunoreactive for SP and NKA in these glands. The present data demonstrate that the major part of the glandular SP and NKA innervation in the minor salivary glands derives from the trigeminal ganglia. The distribution of the peripheral nerve fibers indicates that they may play a role in the delivery of potent neuropeptides involved in the vascular, secretory, and motor (myoepithelial cells) functions of salivary glands.     

Immunoreactive atrial natriuretic polypeptides in the adrenal medulla and sympathetic ganglia

Atrial natriuretic polypeptide (ANP)-like immunoreactivity was found in the rat adrenal gland by using indirect immunofluorescence and peroxidase-antiperoxidase techniques. ANP-like immunostaining was present in most of chromaffin cells with varying degrees of immunoreactivity. The majority of medullary cells displayed very intense immunostaining, and several clusters revealed weaker immunostaining. No staining was found in the adrenal cortex or in the nerve fibers in this organ. In the consecutive sections treated for dopamine-beta-hydroxylase (DBH), apparently all medullary cells had intense immunofluorescence for DBH and its distribution pattern was very similar to that for ANP-like immunoreactivity. While phenylethanolamine N-methyltransferase (PNMT) immunoreactive cells largely corresponded to the intensely stained ANP-like immunoreactive cells, suggesting that adrenaline cells contained a large amount of ANP-like substance, noradrenaline cells contained a smaller amount of this substance than adrenaline cells. Ultrastructural study showed that end-products due to the immunoreaction with the ANP antiserum were primarily associating with chromaffin granules. In addition, the presence of ANP-like immunoreactivity was investigated in several sympathetic ganglia of the rat. No principal ganglion cells were ANP-positive, whereas a few small intensely fluorescent (SIF) cells were ANP-immunoreactive. The present findings suggest that catecholamines coexist with ANP which has a natriuretic and vasodilating effect, in adrenal medullary cells and SIF cells in several rat sympathetic ganglia, but not in principal ganglion cells.     

Muscarinic and nicotinic receptor-mediated Ca2+ dynamics in rat adrenal chromaffin cells during development

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 β-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 β-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 β-hydroxylase and phenylethanolamine N-methyltransferase, whereas some cells were reactive to dopamine β-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.     

Localization of enkephalins in adrenaline cells and the nerves innervating adrenaline cells in rat adrenal medulla

The coexistence of met5- and leu5-enkephalin-like immunoreactivities with catecholamines in the rat adrenal medulla was studied with combined fluorescence microscopy and immunocytochemistry. Both met5- and leu5-enkephalin-like immunoreactivities were localized in few heavily stained adrenaline cells and in a population of nerves innervating adrenaline cells and as well as ganglion cells among the adrenaline cells. Only occasionally single noradrenaline cells exhibited light immunostaining for both enkephalins but no positive fibers could be found around the noradrenaline cells. In electron microscope the immunoreaction was seen in the granules of the adrenaline cells and in the large synaptic vesicles of the nerve terminals around the adrenaline cells. The present findings suggest that enkephalin-like immunoreactivity coexists mainly with adrenaline in rat adrenal medulla and that the enkephalin immunoreactive terminals regulate secretion of adrenaline from rat adrenal medulla.     

Localization of enkephalins in adrenaline cells and the nerves innervating adrenaline cells in rat adrenal medulla

Summary The coexistence of met5- and leu5-enkephalinlike immunoreactivities with catecholamines in the rat adrenal medulla was studied with combined fluorescence microscopy and immunocytochemistry. Both met5- and leu5-enkephalin-like immunoreactivities were localized in few heavily stained adrenaline cells and in a population of nerves innervating adrenaline cells and as well as ganglion cells among the adrenaline cells. Only occasionally single noradrenaline cells exhibited light immunostaining for both enkephalins but no positive fibers could be found around the noradrenaline cells. In electron microscope the immunoreaction was seen in the granules of the adrenaline cells and in the large synaptic vesicles of the nerve terminals around the adrenaline cells. The present findings suggest that enkephalin-like immunoreactivity coexists mainly with adrenaline in rat adrenal medulla and that the enkephalin immunoreactive terminals regulate secretion of adrenaline from rat adrenal medulla.     

Transient involvement of enkephalins in both the sympathetic and parasympathetic innervations of the submandibular gland of rats

Summary A time course study with enkephalin(Enk)-like immunoreactivity has revealed that nerve fibers intensely immunoreactive for Enk-8 appeared transiently only during the postnatal week 2 and 4 within the acini as well as in the inter- and intralobular connective tissues of the submandibular gland of rats. At these stages numerous nerve fibers immunoreactive for tyrosine hydroxylase (TH) appeared also in the inter- and intralobular connective tissues and within the acini. Coincidently with these postnatal stages, abundant Enk-immunoreactive principal ganglion cells appeared in the superior cervical ganglion. These were not immunoreactive for neuropeptide tyrosine (NPY). A substantial number of Enk-immunoreactive ganglion cells were also present in the submandibular ganglia at these younger postnatal stages. Superior cervical ganglionectomy at these stages resulted in a marked decrease in number of the inter- and intralobular Enk-immunoreactive nerve fibers, a slight decrease in number of the intraacinar Enk-immunoreactive nerve fibers, and almost complete disappearance of intraglandular TH-immunoreactive nerve fibers. Immuno-electron-microscopic analysis revealed that Enk-immunoreactive nerve fibers in the submandibular gland were identified as electron-dense neuronal profiles enclosed by Schwann cells in the inter- and intralobular connective tissues and those directly apposed to secretory cells within the acini. They contained small clear vesicles mixed with some large granular vesicles. After postnatal week 6, no Enk-immunoreactive nerve fibers were detected in the submandibular gland, and no TH-immunoreactive nerve fibers were seen within the acini, while TH-immunoreactive nerve fibers remained numerous in the inter- and intralobular connective tissues. These findings indicate that both sympathetic and parasympathetic nerve fibers exhibit Enk-like immunoreactivity transiently during postnatal weeks 2 and 4. It is further indicated that the inter- and intralobular nerve fibers lose Enk-like immunoreactivity while the intraacinar fibers disappear at the adult stage.     

The distribution of cGMP in the adrenal gland in the rat, guinea pig and mouse after stimulation with sodium nitroprusside

Nitric oxide (NO) acts as an intercellular messenger molecule in the nervous system. In the adrenal gland sympathetic preganglionic fibers innervating the medulla, as well as intrinsic neural ganglion cells, contain nitric oxide synthase (NOS). Nitric oxide stimulates the soluble enzyme guanylate cyclase forming cyclic GMP (cGMP). Using sodium nitroprusside (SNP) as nitric oxide donor we have studied the putative target cells for nitric oxide in the rat adrenal gland, both in vivo and in vitro. The guinea pig and a few mouse adrenal glands were studied after SNP perfusion for comparison. Our results show that after vascular perfusion with a high concentration (3 mM) of SNP both noradrenaline and adrenaline chromaffin cells express cGMP-like immunoreactivity in all three species. After incubation of rat adrenal slices with SNP primarily the noradrenaline chromaffin cells are cGMP-positive. In contrast, detectable levels of cGMP-like immunoreactivity were not found in neuronal ganglion cells. In the adrenal cortex cGMP-like immunoreactivity was seen in blood vessel walls, in small cells with processes forming a reticular network, at least partly presumably representing endothelial cells, as well as in some presumable nerve terminals. These findings support the view that chromaffin cells, especially the noradrenergic ones and blood vessels, are targets for nitric oxide in the adrenal gland.     

Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland

Summary The adrenal medulla appears to exert a regulatory influence on adrenocortical steroidogenesis. We have therefore studied the morphology of rat, porcine and bovine adrenals in order to characterize the contact zones of adrenomedullary and adrenocortical tissues. The distribution of chromaffin cells located within the adrenal cortex and of cortical cells located within the adrenal medulla was investigated. Chromaffin cells were characterized by immunostaining for synaptophysin and chromogranin A, both being considered specific for neuroendocrine cells. Cortical cells were characterized by immunostaining for 17-hydroxylase, an enzyme of the steroid pathway. Cellular contacts of chromaffin cells and cortical cells were examined at the electron microscopical level. In rat and porcine adrenals, rays of chromaffin cells, small cell clusters and single chromaffin cells or small invaginations from the medulla could be detected in all three zones of the cortex. Chromaffin cells often spread in the subcapsular space of the zona glomerulosa. In porcine and bovine adrenals, 17-hydroxylase immunoreactive cells were localized within the medulla. Single cortical cells and small accumulations of cells were spread throughout this region. At the ultrastructural level, the chromaffin cells located within the cortex in pig and rat adrenals formed close cellular contacts with cortical cells in all three zones. Our morphological data provide evidence for a possible paracrine role of chromaffin cells; this may be important for the neuroregulation of the adrenal cortex.     

Protein kinase c-β-like immunoreactivity in the developing and adult rat adrenal gland

Summary Protein kinase c--like immunoreactivity was studied in the adrenal gland of adult rats and at different pre- and postnatal stages of development (E17-P21) with an antibody specific to both the ₂₁ and - subtypes of the kinase. In the adult rat adrenal gland, the immunoreactivity was seen in numerous nerve fibres in the adrenal medulla both in bundles and individually forming occasionally dense networks around chromaffin cell groups. Several protein kinase c--immunoreactive fibres were also observed transversing the adrenal cortex towards the medulla. No remaining immunoreactive fibres two weeks after transection of the splanchnic nerve could be seen; nor was any immunoreactivity observed in the chromaffin cells of the adrenal medulla or in the cortical cells, but some faintly immunoreactive ganglion cells were detected in the adrenal medulla. The amount and distribution of protein kinase c--like immunoreactivity in the fetal and developing adrenals was very similar to that seen in the adrenal glands of adult rats. On the basis of its localization, the -subtype of protein kinase c does not appear to be directly involved in the release of catecholamines from the adrenal medulla, but it might have a role in the regulation of neurotransmitter release from preganglionic cholinergic neurons.     

Distribution and colocalization of nitric oxide synthase and NADPH-diaphorase in adrenal gland of developing,adult and aging Sprague-Dawley rats

The distribution and colocalization of nitric oxide synthase and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase) was investigated in the adrenal gland of developing, adult and aging rats with the use of immunohistochemical and histochemical techniques. Nitric oxide synthase-immunoreactive neurons within the adrenal gland were found from the 20th day of gestation onwards. During early development the neurons were found as small clusters of smaller-size cells compared to those observed in the adult gland. Their number reached that of adult level by the 4th day after birth, and in the glands from aging rats a 28.6% increase was observed. Whilst no immunofluorescence was seen in chromaffin cells during early development, some cells from glands of aging rats showed nitric oxide synthase-immunoreactivity with varying intensity. The immunoreactive neurons from postnatal rat adrenals were also positive for NADPH-diaphorase, whilst those in prenatal rats were negative or lightly stained. Nitric oxide synthase-immunoreactive nerve fibres were present in all adrenal glands examined from the 16th day of gestation onwards. A considerable degree of variation in the distribution of immunoreactive fibres both in medulla and outer region of cortex at the different age groups was observed and described. Most, but not all, nitric oxide synthase-immunoreactive nerve fibres also showed NADPH-diaphorase staining.     

Substance P-containing axon terminals in the mucosa of the human urinary bladder: pre-embedding immunohistochemistry using cryostat sections for electron microscopy

The ultrastructure of substance P (SP)-containing axon terminals in the mucosa of the human urinary bladder was studied. Numerous SP-immunoreactive varicose nerve fibers were seen in the lamina propria, and most of them ran freely in the connective tissue. Many SP-immunoreactive nerve fibers were observed beneath the epithelium, and perivascular SP-immunoreactive nerves were also found in the submucosal layer. We observed a total of 305 SP-immunoreactive (IR) axon terminals, of which most (89.6%) were free nerve endings at the ultrastructural level; the rest of the SR-IR axon terminale were seen in the vicinity of the epithelium and blood vessels in the lamina propria. Varicose regions of SP-IR axon terminals contained large granular and small agranular synaptic vesicles, and most of them partially lacked a Schwann cell sheath. In some SP-IR varicosities, synaptic vesicles were concentrated in the region without any Schwann cell sheath. Long storage (for more than 1 month) of fixed-tissue pieces in sucrose before freezing has improved the ultrastructure of cryostat sections in pre-embedding immunohistochemistry. Trypsin digestion for the purpose of exposing antigenic sites was also employed before applying the first antiserum.     

VIP like immunoreactive nerves in human respiratory tract. Light and electron microscopic study

The present study provides light and electron microscopical evidence of Vasoactive Intestinal Peptide - (VIP) like immunoreactive nerves in human lower respiratory tract. Peroxidase antiperoxidase (PAP) technique was used to localize VIP-like immunoreactivity light microscopically and ultrastructurally. Under light microscopy, VIP-like immunoreactive nerves were observed in the smooth muscle layer of secondary bronchi to small bronchioli, and in bronchial glands. In addition, positive immunoreactive nervous network to VIP was found around nerve cell bodies in small microganglia. The bronchial epithelium of airway tract did not receive any VIP positive nerve fibers. Ultrastructurally VIP-like positive immunoreaction was localized in large granular vesicles ranging from 90 to 210 nm. Usually VIP-like positive immunoreactive nerve profiles contained several immunoreactive large vesicles (100-210). However, nerve profiles containing only a few positive large vesicles (80-150) were also observed. Under electron microscopy VIP-positive nerve profiles corresponded ultrastructurally to nerve profiles containing large granular vesicles observed in conventional electronmicroscopy. The present study provides new information about the innervation of human lower airway tract and widens the concept of their functional regulation on the anatomical basis reported here.